Wiki FKKT - User contributions [en]

2015-bionano-seminar

2015-06-01T07:58:07Z

ValterB:

= Bionanotehnologija- seminar =
doc. dr. Gregor Gunčar, K2.022

== Seznam seminarjev ==
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Avtor 1'''
| align="center" style="background:#f0f0f0;"|'''Avtor 2'''
| align="center" style="background:#f0f0f0;"|'''Naslov seminarja'''
| align="center" style="background:#f0f0f0;"|'''Datum za oddajo'''
| align="center" style="background:#f0f0f0;"|'''Datum predstavitve'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 1'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 2'''
|-
| Anže Prašnikar||Monika Praznik ||||29.03.||31.03.||Aneja Tuljak||Angelika Vižintin
|-
| Varja Božič||Eva Knapič||Razgradljivi kondomi s protimikrobno zaščito||29.03.||31.03.||Eva Udovič||Maja Grdadolnik
|-
| Belkisa Velagić||Aleksander Benčič||Avtomobilski encimski katalizator||29.03.||31.03.||Nika Kurinčič||Tjaša Goričan
|-
| Naja Vrankar||Valter Bergant||Modifikacija črevesne mikroflore pri debelosti||31.03.||02.04.||Nataša Žigante||Luka Smole
|-
| Tilen Volčanšek||Veronika Jarc||||31.03.||02.04.||Anže Prašnikar||Jakob Gašper Lavrenčič
|-
| Tanja Lipec||Iza Ogris||Test občutljivosti na gluten z neprebavljivo kapsulo||31.03.||02.04.||Varja Božič||Klara Tereza Novoselc
|-
| Katja Lovrin||Mitja Crček||Uporaba nitrifikacijskih encimov pri kmetovanju||05.04.||07.04.||Belkisa Velagić||Monika Praznik
|-
| Saša Balažic||Urban Javoršek ||||05.04.||07.04.||Naja Vrankar||Eva Knapič
|-
| Urban Borštnik||Sara Primec||||05.04.||07.04.||Tilen Volčanšek||Aleksander Benčič
|-
| Nives Ahlin||Kim Kos||||12.04.||14.04.||Tanja Lipec||Valter Bergant
|-
| Matic Bevec||Estera Merljak||Antimaček®||12.04.||14.04.||Katja Lovrin||Veronika Jarc
|-
| Vida Špindler||Jernej Pušnik||||12.04.||14.04.||Saša Balažic||Iza Ogris
|-
| Jasmina Sedmak||Maxi Sagmeister||Brezžični zobni nanobiosenzor ||19.04.||21.04.||Urban Borštnik||Mitja Crček
|-
| Sanja Popović||Benjamin Bajželj||||19.04.||21.04.||Nives Ahlin||Urban Javoršek
|-
| Blaž Komar||Alja Zottel||||19.04.||21.04.||Matic Bevec||Sara Primec
|-
| Blaž Perič||Katarina Uršič||Biorazgradljivi žvečilni gumi z antibakterijskimi lastnostmi||03.05.||05.05.||Simon Preložnik||Kim Kos
|-
| Simon Preložnik||Maja Remškar||Preprost dostavni sistem za omega-3 maščobne kisline||03.05.||05.05.||Jasmina Sedmak||Estera Merljak
|-
| Aneja Tuljak||Tina Gregorič||Stekleničke z biosenzorjem za detekcijo ''E.coli''||03.05.||05.05.||Sanja Popović||Jernej Pušnik
|-
| Damir Hamulić||Anita Kustec||Pretvorba CO2 v uporabne proizvode s pomočjo umetne fotosinteze||10.05.||12.05.||Blaž Komar||Maxi Sagmeister
|-
| Janja Fortin||Tina Snoj|| Zaviralec nadležnih posledic komarjevega pika (Antikomarin) ||10.05.||12.05.||Blaž Perič||Benjamin Bajželj
|-
| Rajko Vnuk||Mojca Banič||||10.05.||12.05.||Vida Špindler||Alja Zottel
|-
| Rok Grm||Ajda Rojc||||17.05.||19.05.||Kaja Javoršek||Katarina Uršič
|-
| Kristina Gavranić||Barbara Žužek||Protimikrobni žvečilni gumi s hidroksiapatitnimi nanodelci za remineralizacijo zobne sklenine||17.05.||19.05.||Damir Hamulić||Maja Remškar
|-
| Urška Mohorič||Griša Prinčič||Verižna reakcija s polimerazo na osnovi denaturacije z magnetnimi nanodelci||17.05.||19.05.||Janja Fortin||Tina Gregorič
|-
| Maja Ramić||Nejc Petrišič||||24.05.||26.05.||Rajko Vnuk||Anita Kustec
|-
| Barbara Jeras||Tamara Marić||||24.05.||26.05.||Rok Grm||Tina Snoj
|-
| Matic Urlep||Samo Zakotnik||Uporaba optogenetike za lajšanje simptomov Parkinsonove bolezni||24.05.||26.05.||Kristina Gavranić||Mojca Banič
|-
| Urban Verbič||Angelika Vižintin||||31.05.||02.06.||Urška Mohorič||Ajda Rojc
|-
| Nataša Žigante||Maja Grdadolnik||||31.05.||02.06.||Maja Ramić||Barbara Žužek
|-
| Kaja Javoršek||Tjaša Goričan||Kontaktne leče, ki preventivno ščitijo pred očesnimi boleznimi (proti slepoti)||31.05.||02.06.||Barbara Jeras||Griša Prinčič
|-
| Eva Udovič||Luka Smole||||07.06.||09.06.||Matic Urlep||Nejc Petrišič
|-
| Nika Kurinčič||Jakob Gašper Lavrenčič||||07.06.||09.06.||Urban Verbič||Tamara Marić
|-
| Klara Tereza Novoselc||||||07.06.||09.06.||Samo Zakotnik||
|}

== Gradivo za predavanja ==
Gradivo za predavanja najdete v [http://ucilnica.fkkt.uni-lj.si/ spletni učilnici].

==Naloga==
'''Vaša naloga je: '''
Po dva študenta skupaj pripravita projektno nalogo iz področja Bionanotehnologije. Najpomembnejša je originalna ideja za nek izvedljiv projekt.
Predlagana struktura:
* Uvod
* Predstavitev problema, znanstvena izhodišča, cilji
* Izvedba projekta, metodologija, tehnike, materiali, vprašanja, hipoteze
* Literatura

Za pripravo seminarja velja naslednje: 
* Prva stran seminarja naj vsebuje naslov projekta, avtorje, povzetek (od 130 do 160 besed) in grafični povzetek (čez približno pol strani)
* Seminar pripravite v obliki seminarske naloge na ~5 straneh A4 (pisava 12, enojni razmak, 2,5 cm robovi). Zelo pomembno je, da je obseg od 1500 do 2000 besed . Seminarska naloga mora vsebovati najmanj tri slike. Slika mora imeti legendo in v besedilu mora biti na ustreznem mestu sklic na sliko. 
* Seminar oddajte do datuma oddaje, ki je naveden v tabeli v elektronski obliki z uporabo [http://bio.ijs.si/~zajec/poslji/ tega obrazca].
* Vsi seminarji so v elektronski obliki dostopni [http://bio.ijs.si/~zajec/poslji/bioseminar/ tukaj].
* Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo 20 minut, predstavitev pa ne sme biti krajša od 15 minut (popust :-)). Nalogo predstavita oba študenta (razdelita si čas). Recenzenti morajo biti na predstavitvi prisotni.
* Predstavitvi sledi razprava. Recenzenti podajo pripombe k projektu in postavijo po dve vprašanji.
* Na dan predstavitve morate docentu še pred predstavitvijo oddati končno verzijo seminarja v enem izvodu, elektronsko verzijo seminarja in predstavitev pa oddati na strežnik na dan predstavitve do polnoči.

==Imena datotek==
Prosim vas, da vse datoteke poimenujete po naslednjem receptu:
* 19_nano_Priimek1_Priimek2.doc(x) za seminar, npr. 19_nano_Craik_Venter.docx
* 19_nano_Priimek1_Priimek2.ppt(x) za prezentacijo, npr. 19_nano_Craik_Venter.pptx

==Ocenjevanje seminarjev==
Recenzenti ocenijo seminar tako, da izpolnijo [https://docs.google.com/forms/d/1WdCXoXo1zkRrVlLKIcEV1z_MyhavU-3ERBm9n2oiawI/viewform recenzentsko poročilo] na spletu. Recenzentsko poročilo morate oddati najkasneje do predstavitve seminarja.

== Mnenje o predstavitvi ==
Vsak posameznik '''mora''' oceniti seminar, tako da odda svoje [https://docs.google.com/forms/d/1ToLPn78T9W3G6Hm5hV0mLseFYghiLQMlRPGb0J5zft8/viewform mnenje] najkasneje v sedmih dneh po predstavitvi. Kdor na seminarju ni bil prisoten, mnenja '''ne sme''' oddati.
Na [http://bit.ly/bntmnenja tej strani] lahko preverite, če ste svoje mnenje za določen seminar že oddali.

==Urejanje spletnih strani na wikiju==
Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''

==Citiranje virov==
Citiranje je možno po več shemah, važno je, da se držite ene same. V seminarskih nalogah in diplomskih nalogah FKKT uprabljajte shemo citiranja, ki je pobarvana zeleno.
Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.
Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.zveza-zotks.si/gzm/dokumenti/literatura.html Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati. 
'''Citiranje knjig:''' 
Priimek, I. ''Naslov''. Kraj: Založba, letnica. 
Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN. 

Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005. 
Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4. 
Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.).

'''Citiranje člankov:''' 
Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani. 
Lartigue, C., Glass, J. I., Alperovich, N., Pieper, R., Parmar, P. P., Hutchison III, C. A., Smith, H. O. in Venter, J. C.
Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, 317, str. 632-638.

Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole: 
Priimek, I. (letnica, številka). Naslov. Naslov revije, strani. 
Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.

Revija Science uporablja skrajšani zapis: 
C. Lartigue ''et al''. Science 317, 632 (2007) 

V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne). Navesti morate tudi vse avtorje dela, razen v primeru, ko jih je 10 ali več. Takrat navedite le prvih devet, za ostale pa uporabite okrajšavo in sod. (in sodelavci). Pred zadnjim avtorjem naj bo vedno besedica "in".

'''Citiranje spletnih virov:''' 
Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov 
strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life 
Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.

A synthetic oscillatory network of transcriptional regulators

2015-01-04T09:55:23Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking, which helped jumpstart the aforementioned science.

== Overview ==

The artificial oscillatory system encoded on a plasmid will be inserted into the pool of host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose ''Escherichia coli'', the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor, which acts as a part of oscillator, or induced or leaked expression of endogenous LacI repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list is a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. At this point reader is encouraged to fully understand the cyclical behaviour of the represillator, as the concept is a vital part of this chapter. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors. These phenomena are explained in more detail in the following paragraphs.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Such properties move the biological system closer to a theoretical binary oscillator. In such a device, all components have only two valid output states, 0 or 1. In represillator terms, the expression of all repressor components would therefore be either maximal or zero. This would leave no room for a stable steady-state in a configuration using an odd number of components - like three, used in the represillator. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they combined λ PL promoter with lac and tet operator sequences producing hybrid versions, which are stronger yet still tightly repressible, termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or the list above as required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage; it already contains operator sites for the lambda cI protein that represses the promoter. Additionally, they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes - close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell. Low enough half-life of involved proteins is paramount for oscillations to be transferred to protein stage with little phase delay. If life-span of protein is short compared to the peak-to-peak time of oscillation, proteins synthesized at the time of the peak will clear from the cell quickly and therefore enhance the dynamics of the system by being in-phase with the transcriptional oscillations.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed the so called reporter construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator system. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour, over 40 % of the observed cells were shown to follow such regime. Despite that, the noise in individual cells due to stochastic effects, coupled with inability of cells to synchronize oscillations across population, caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Attempts at synchronisation of cell population oscillators using IPTG were unsuccessful. Because of such effects, the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it, the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30 °C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause various effects, such as changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article, authors contemplate the theoretical basis behind the work described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation of natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance, the authors ask whether similar synthetic circuits would retain this highly valued property and mention it as possible logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

----

References:

Michael B. Elowitz & Stanislas Leibler, A synthetic oscillatory network of transcriptional regulators, Letters to Nature, 2010 and references therein.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-04T09:45:46Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking, which helped jumpstart the aforementioned science.

== Overview ==

The artificial oscillatory system encoded on a plasmid will be inserted into the pool of host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose ''Escherichia coli'', the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor, which acts as a part of oscillator, or induced or leaked expression of endogenous LacI repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list is a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. At this point reader is encouraged to fully understand the cyclical behaviour of the represillator, as the concept is a vital part of this chapter. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors. These phenomena are explained in more detail in the following paragraphs.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Such properties move the biological system closer to a theoretical binary oscillator. In such a device, all components have only two valid output states, 0 or 1. In represillator terms, the expression of all repressor components would therefore be either maximal or zero. This would leave no room for a stable steady-state in a configuration using an odd number of components - like three, used in the represillator. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they combined λ PL promoter with lac and tet operator sequences producing hybrid versions, which are stronger yet still tightly repressible, termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or the list above as required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage; it already contains operator sites for the lambda cI protein that represses the promoter. Additionally, they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes - close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell. Low enough half-life of involved proteins is paramount for oscillations to be transferred to protein stage with little phase delay. If life-span of protein is short compared to the peak-to-peak time of oscillation, proteins synthesized at the time of the peak will clear from the cell quickly and therefore enhance the dynamics of the system by being in-phase with the transcriptional oscillations.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed the so called reporter construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator system. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

----

References:

Michael B. Elowitz & Stanislas Leibler, A synthetic oscillatory network of transcriptional regulators, Letters to Nature, 2010 and references therein.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-04T09:43:26Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking, which helped jumpstart the aforementioned science.

== Overview ==

The artificial oscillatory system encoded on a plasmid will be inserted into the pool of host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose ''Escherichia coli'', the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor, which acts as a part of oscillator, or induced or leaked expression of endogenous LacI repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list is a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. At this point reader is encouraged to fully understand the cyclical behaviour of the represillator, as the concept is a vital part of this chapter. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors. These phenomena are explained in more detail in the following paragraphs.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Such properties move the biological system closer to a theoretical binary oscillator. In such a device, all components have only two valid output states, 0 or 1. In represillator terms, the expression of all repressor components would therefore be either maximal or zero. This would leave no room for a stable steady-state in a configuration using an odd number of components - like three, used in the represillator. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they combined λ PL promoter with lac and tet operator sequences producing hybrid versions, which are stronger yet still tightly repressible, termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or the list above as required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage; it already contains operator sites for the lambda cI protein that represses the promoter. Additionally, they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes - close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell. Low enough half-life of involved proteins is paramount for oscillations to be transferred to protein stage with little phase delay. If life-span of protein is short compared to the peak-to-peak time of oscillation, proteins synthesized at the time of the peak will clear from the cell quickly and therefore enhance the dynamics of the system by being in-phase with the transcriptional oscillations.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

----

References:

Michael B. Elowitz & Stanislas Leibler, A synthetic oscillatory network of transcriptional regulators, Letters to Nature, 2010 and references therein.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-04T09:34:10Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking, which helped jumpstart the aforementioned science.

== Overview ==

The artificial oscillatory system encoded on a plasmid will be inserted into the pool of host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose ''Escherichia coli'', the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor, which acts as a part of oscillator, or induced or leaked expression of endogenous LacI repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list is a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. At this point reader is encouraged to fully understand the cyclical behaviour of the represillator, as the concept is a vital part of this chapter. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors. These phenomena are explained in more detail in the following paragraphs.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Such properties move the biological system closer to a theoretical binary oscillator. In such a device, all components have only two valid output states, 0 or 1. In represillator terms, the expression of all repressor components would therefore be either maximal or zero. This would leave no room for a stable steady-state in a configuration using an odd number of components - like three, used in the represillator. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they combined λ PL promoter with lac and tet operator sequences producing hybrid versions, which are stronger yet still tightly repressible, termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or the list above as required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage; it already contains operator sites for the lambda cI protein that represses the promoter. Additionally, they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes - close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

----

References:

Michael B. Elowitz & Stanislas Leibler, A synthetic oscillatory network of transcriptional regulators, Letters to Nature, 2010 and references therein.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-04T09:32:43Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking, which helped jumpstart the aforementioned science.

== Overview ==

The artificial oscillatory system encoded on a plasmid will be inserted into the pool of host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose ''Escherichia coli'', the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor, which acts as a part of oscillator, or induced or leaked expression of endogenous LacI repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list is a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. At this point reader is encouraged to fully understand the cyclical behaviour of the represillator, as the concept is a vital part of this chapter. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors. These phenomena are explained in more detail in the following paragraphs.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Such properties move the biological system closer to a theoretical binary oscillator. In such a device, all components have only two valid output states, 0 or 1. In represillator terms, the expression of all repressor components would therefore be either maximal or zero. This would leave no room for a stable steady-state in a configuration using an odd number of components - like three, used in the represillator. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they combined λ PL promoter with lac and tet operator sequences producing hybrid versions, which are stronger yet still tightly repressible, termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or the list above as required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage; it already contains operator sites for the lambda cI protein that represses the promoter. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

----

References:

Michael B. Elowitz & Stanislas Leibler, A synthetic oscillatory network of transcriptional regulators, Letters to Nature, 2010 and references therein.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-04T09:29:32Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking, which helped jumpstart the aforementioned science.

== Overview ==

The artificial oscillatory system encoded on a plasmid will be inserted into the pool of host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose ''Escherichia coli'', the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor, which acts as a part of oscillator, or induced or leaked expression of endogenous LacI repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list is a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. At this point reader is encouraged to fully understand the cyclical behaviour of the represillator, as the concept is a vital part of this chapter. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors. These phenomena are explained in more detail in the following paragraphs.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Such properties move the biological system closer to a theoretical binary oscillator. In such a device, all components have only two valid output states, 0 or 1. In represillator terms, the expression of all repressor components would therefore be either maximal or zero. This would leave no room for a stable steady-state in a configuration using an odd number of components - like three, used in the represillator. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they combined λ PL promoter with lac and tet operator sequences producing hybrid versions, which are stronger yet still tightly repressible, termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or the list above as required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

----

References:

Michael B. Elowitz & Stanislas Leibler, A synthetic oscillatory network of transcriptional regulators, Letters to Nature, 2010 and references therein.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-04T09:24:59Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking, which helped jumpstart the aforementioned science.

== Overview ==

The artificial oscillatory system encoded on a plasmid will be inserted into the pool of host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose ''Escherichia coli'', the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor, which acts as a part of oscillator, or induced or leaked expression of endogenous LacI repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list is a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. At this point reader is encouraged to fully understand the cyclical behaviour of the represillator, as the concept is a vital part of this chapter. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors. These phenomena are explained in more detail in the following paragraphs.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Such properties move the biological system closer to a theoretical binary oscillator. In such a device, all components have only two valid output states, 0 or 1. In represillator terms, the expression of all repressor components would therefore be either maximal or zero. This would leave no room for a stable steady-state in a configuration using an odd number of components - like three, used in the represillator. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

----

References:

Michael B. Elowitz & Stanislas Leibler, A synthetic oscillatory network of transcriptional regulators, Letters to Nature, 2010 and references therein.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-04T09:18:45Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking, which helped jumpstart the aforementioned science.

== Overview ==

The artificial oscillatory system encoded on a plasmid will be inserted into the pool of host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose ''Escherichia coli'', the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor, which acts as a part of oscillator, or induced or leaked expression of endogenous LacI repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list is a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. At this point reader is encouraged to fully understand the cyclical behaviour of the represillator, as the concept is a vital part of this chapter. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors. These phenomena are explained in more detail in the following paragraphs.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

----

References:

Michael B. Elowitz & Stanislas Leibler, A synthetic oscillatory network of transcriptional regulators, Letters to Nature, 2010 and references therein.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-04T09:14:59Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking, which helped jumpstart the aforementioned science.

== Overview ==

The artificial oscillatory system encoded on a plasmid will be inserted into the pool of host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose ''Escherichia coli'', the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor, which acts as a part of oscillator, or induced or leaked expression of endogenous LacI repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list is a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

----

References:

Michael B. Elowitz & Stanislas Leibler, A synthetic oscillatory network of transcriptional regulators, Letters to Nature, 2010 and references therein.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-04T09:12:27Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking, which helped jumpstart the aforementioned science.

== Overview ==

The artificial oscillatory system encoded on a plasmid will be inserted into the pool of host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose ''Escherichia coli'', the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor, which acts as a part of oscillator, or induced or leaked expression of endogenous LacI repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list represents a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

----

References:

Michael B. Elowitz & Stanislas Leibler, A synthetic oscillatory network of transcriptional regulators, Letters to Nature, 2010 and references therein.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-04T09:11:09Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking, which helped jumpstart the aforementioned science.

== Overview ==

The artificial oscillatory system encoded on a plasmid will be inserted into the pool of host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose ''Escherichia coli'', the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor, which acts as a part of oscillator, or induced or leaked expression of endogenous LacI repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing. Great efforts were made, cross my heart, by student deep in the study of synthetic biology to write in an understanding manner, but sometimes one gets sloppy with the language for which most sincere apologies should suffice.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list represents a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

----

References:

Michael B. Elowitz & Stanislas Leibler, A synthetic oscillatory network of transcriptional regulators, Letters to Nature, 2010 and references therein.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-04T09:09:26Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking, which helped jumpstart the aforementioned science.

== Overview ==

The artificial oscillatory system encoded on a plasmid will be inserted into the pool of host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose ''Escherichia coli'', the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor or induced or leaked expression of endogenous repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing. Great efforts were made, cross my heart, by student deep in the study of synthetic biology to write in an understanding manner, but sometimes one gets sloppy with the language for which most sincere apologies should suffice.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list represents a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

----

References:

Michael B. Elowitz & Stanislas Leibler, A synthetic oscillatory network of transcriptional regulators, Letters to Nature, 2010 and references therein.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-04T09:08:52Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking, which helped jumpstart the aforementioned science.

== Overview ==

The artificial oscillatory system encoded on a plasmid will be inserted into the pool of host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose Escherichia coli, the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor or induced or leaked expression of endogenous repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing. Great efforts were made, cross my heart, by student deep in the study of synthetic biology to write in an understanding manner, but sometimes one gets sloppy with the language for which most sincere apologies should suffice.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list represents a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

----

References:

Michael B. Elowitz & Stanislas Leibler, A synthetic oscillatory network of transcriptional regulators, Letters to Nature, 2010 and references therein.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-04T09:07:11Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking, which helped jumpstart the aforementioned science.

== Overview ==

The artificial oscillatory system, designed by the authors, will be inserted into host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose Escherichia coli, the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor or induced or leaked expression of endogenous repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing. Great efforts were made, cross my heart, by student deep in the study of synthetic biology to write in an understanding manner, but sometimes one gets sloppy with the language for which most sincere apologies should suffice.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list represents a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

----

References:

Michael B. Elowitz & Stanislas Leibler, A synthetic oscillatory network of transcriptional regulators, Letters to Nature, 2010 and references therein.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-03T17:52:14Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking.

== Overview ==

The artificial oscillatory system, designed by the authors, will be inserted into host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose Escherichia coli, the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor or induced or leaked expression of endogenous repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing. Great efforts were made, cross my heart, by student deep in the study of synthetic biology to write in an understanding manner, but sometimes one gets sloppy with the language for which most sincere apologies should suffice.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list represents a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

----

References:

Michael B. Elowitz & Stanislas Leibler, A synthetic oscillatory network of transcriptional regulators, Letters to Nature, 2010 and references therein.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-03T17:51:55Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking.

== Overview ==

The artificial oscillatory system, designed by the authors, will be inserted into host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose Escherichia coli, the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor or induced or leaked expression of endogenous repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing. Great efforts were made, cross my heart, by student deep in the study of synthetic biology to write in an understanding manner, but sometimes one gets sloppy with the language for which most sincere apologies should suffice.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list represents a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

----

References:

Michael B. Elowitz & Stanislas Leibler,A synthetic oscillatory network of transcriptional regulators, Letters to Nature, 2010 and references therein.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-03T17:37:34Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an ''in silico'' model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking.

== Overview ==

The artificial oscillatory system, designed by the authors, will be inserted into host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose Escherichia coli, the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, ''E. coli'' regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host ''E. coli'' should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full ''lac'' operon; this removes the interference of additional binding sites for LacI repressor or induced or leaked expression of endogenous repressor protein. Other parts used in the design of the represillator are not found in ''E. coli'' genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing. Great efforts were made, cross my heart, by student deep in the study of synthetic biology to write in an understanding manner, but sometimes one gets sloppy with the language for which most sincere apologies should suffice.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list represents a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-03T17:35:19Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an in silico model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking.

== Overview ==

The artificial oscillatory system, designed by the authors, will be inserted into host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose Escherichia coli, the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, E. coli regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host E. coli should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full lac operon; this removes the interference of additional binding sites for LacI repressor or induced or leaked expression of endogenous repressor protein. Other parts used in the design of the represillator are not found in E. coli genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing. Great efforts were made, cross my heart, by student deep in the study of synthetic biology to write in an understanding manner, but sometimes one gets sloppy with the language for which most sincere apologies should suffice.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list represents a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in E. coli) by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into E. coli. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect E. coli cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

[[SB students resources]]

A synthetic oscillatory network of transcriptional regulators

2015-01-03T17:34:09Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an in silico model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking.

== Overview ==

The artificial oscillatory system, designed by the authors, will be inserted into host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose Escherichia coli, the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, E. coli regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host E. coli should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full lac operon; this removes the interference of additional binding sites for LacI repressor or induced or leaked expression of endogenous repressor protein. Other parts used in the design of the represillator are not found in E. coli genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing. Great efforts were made, cross my heart, by student deep in the study of synthetic biology to write in an understanding manner, but sometimes one gets sloppy with the language for which most sincere apologies should suffice.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list represents a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in E. coli) by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into E. coli. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect E. coli cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

[[SB students resources]]

SB students resources

2015-01-03T17:33:32Z

ValterB:

===Introduction to our students resources in Synthetic Biology===
(Marko Dolinar)

Synthetic biology made a vast progress in good 10 years since it established itself as an interdisciplinary field of research on the interface of molecular biology and engineering. University of Ljubljana Faculty of Chemistry and Chemical Technology has introduced a Synthetic Biology course as a part od Biochemistry MSc programme only in 2013/14. This is relatively late, considering a great success of Slovenian students at iGEM competitions since their first attendance in 2006. On the other hand, the field is still in its first stages if development and a complete textbook for a MSc level course is still missing. This is the reason why our students collaborated on the preparation of a Synthetic Biology textbook with the working title Synthetic Biology - A Students Textbook. It exists as a draft that is not publicly available and is actually part 1 of a (to be) 2-volumes title. Part I is subtitled Engineering Biology, while Part II (that currently doesn't exisist yet) will be subtitled Synthetic Biology Applications.

As in all highly competitive fields of science and technology, students should be following recent progress by reading articles in high quality journals. However, this is often a very difficult task, especially at the BSc level. Specificities of the scientific and technical language, push of publishers towards very short methodological chapters and limited knowledge studens might have about advanced techniques make understanding papers a very challenging task. Therefore, I decided to face MSc students with the challenge to explain selected SB articles in a manner that would make the content of these articles understandable to BSc level students and non-experts.

In 2014/15, seminars in Synthetic Biology include explanations and presentations of some of the top-cited articles from the field of Synthetic Biology. I compiled a list of 95 articles published between 2000 and 2014 having the highest number of citations according to the Web of Science database. The list ends with the paper just exceeding the 100 citations limit. Not included in the list were reviews. With 20 students enrolled in the course, the list has been further reduced to top 40 papers in the field. Students have been asked to check for content (they further eliminated 3 papers which proved to be reviews) and availabitly (they all seemed to be available as full texts with our university subscriptions). My suggestion was to avoid selecting for presentation papers with very similar content. Especially in the field of genome editing there has been a very rapid progress in the past few years resulting in a number of highly-cited articles which could appear very similar in content for a non-specialist. From the shortlist of 37 articles, students selected a topic they believed would be most interesting or easiest to explain. Presentations Will be both written (in English, which is not the mother tongue of my students) and oral (in Slovenian, to establish and maintain Slovenian terminology in the field).

===List of articles for presentation===

This is the list of top-cited papers from the broader field of Synthetic Biology that students chose for explanation in 2014/15 (sorted by year of publication):

#[[A synthetic oscillatory network of transcriptional regulators]], Michael B. Elowitz & Stanislas Leibler, Letters to Nature, 2000 - Valter Bergant
#[[Construction of a genetic toggle switch in Escherichia coli]]. Gardner ''et al''., Nature, 2000 - Urban Bezeljak
#Positive feedback in eukaryotic gene networks: cell differentiation by graded to binary response conversion (2001) - Andreja Bratovš
#Chemical synthesis of poliovirus cDNA: Generation of infectious virus in the absence of natural template (2002) - Veronika Jarc
#Combinatorial synthesis of genetic networks (2002) - Maja Remškar
#Engineering a mevalonate pathway in Escherichia coli for production of terpenoids (2003) - Ana Kapraljević
#Programmed population control by cell-cell communication and regulated killing (2004) - Alja Zottel
#Gene regulation at the single-cell level (2005) - Katarina Uršič
#A synthetic multicellular system for programmed pattern formation (2005) - Mitja Crček
#Long-term monitoring of bacteria undergoing programmed population control in a microchemostat (2005) - Jana Verbančič
#Tuning genetic control through promoter engineering (2005) - Špela Pohleven
#Production of the antimalarial drug precursor artemisinic acid in engineered yeast (2006) - Živa Marsetič
#An improved zinc-finger nuclease architecture for highly specific genome editing (2007) - Eva Knapič
#Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases (2008) - Tamara Marić
#Synthetic protein scaffolds provide modular control over metabolic flux (2009) - Ana Dolinar
#Creation of a bacterial cell controlled by a chemically synthesized genome (2010) Eva Lucija Kozak
#A TALE nuclease architecture for efficient genome editing (2011) Jernej Mustar
#Multiplex genome engineering using CRISPR/Cas systems (2013) - Uroš Stupar
#RNA-guided human genome engineering via Cas9 (2013) - Luka Smole
#One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering (2013) - Andrej Vrankar

''Please link the title of each paper with your written seminar wiki page. Expand the citation according to the following example:
''
#Emergent bistability by a growth-modulating positive feedback circuit. Tan et al., Nature Chem. Biol., 2009

A synthetic oscillatory network of transcriptional regulators

2015-01-03T17:32:13Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an in silico model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking.

== Overview ==

The artificial oscillatory system, designed by the authors, will be inserted into host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose Escherichia coli, the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, E. coli regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host E. coli should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full lac operon; this removes the interference of additional binding sites for LacI repressor or induced or leaked expression of endogenous repressor protein. Other parts used in the design of the represillator are not found in E. coli genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing. Great efforts were made, cross my heart, by student deep in the study of synthetic biology to write in an understanding manner, but sometimes one gets sloppy with the language for which most sincere apologies should suffice.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list represents a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in E. coli) by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into E. coli. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect E. coli cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

[http://wiki.fkkt.uni-lj.si/index.php/SB_students_resources Back to SB student resources]

A synthetic oscillatory network of transcriptional regulators

2015-01-03T17:28:45Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an in silico model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking.

== Overview ==

The artificial oscillatory system, designed by the authors, will be inserted into host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose Escherichia coli, the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, E. coli regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host E. coli should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full lac operon; this removes the interference of additional binding sites for LacI repressor or induced or leaked expression of endogenous repressor protein. Other parts used in the design of the represillator are not found in E. coli genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing. Great efforts were made, cross my heart, by student deep in the study of synthetic biology to write in an understanding manner, but sometimes one gets sloppy with the language for which most sincere apologies should suffice.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)

• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)

• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list represents a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in E. coli) by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into E. coli. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect E. coli cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

A synthetic oscillatory network of transcriptional regulators

2015-01-03T17:25:36Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an in silico model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking.

== Overview ==

The artificial oscillatory system, designed by the authors, will be inserted into host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose Escherichia coli, the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, E. coli regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host E. coli should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full lac operon; this removes the interference of additional binding sites for LacI repressor or induced or leaked expression of endogenous repressor protein. Other parts used in the design of the represillator are not found in E. coli genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing. Great efforts were made, cross my heart, by student deep in the study of synthetic biology to write in an understanding manner, but sometimes one gets sloppy with the language for which most sincere apologies should suffice.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)
• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)
• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list represents a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in E. coli) by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into E. coli. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect E. coli cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

A synthetic oscillatory network of transcriptional regulators

2015-01-03T17:25:12Z

ValterB:

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler

Departments of Molecular Biology and Physics, Princeton University, Princeton,

New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an in silico model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking.

== Overview ==

The artificial oscillatory system, designed by the authors, will be inserted into host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose Escherichia coli, the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, E. coli regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host E. coli should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full lac operon; this removes the interference of additional binding sites for LacI repressor or induced or leaked expression of endogenous repressor protein. Other parts used in the design of the represillator are not found in E. coli genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing. Great efforts were made, cross my heart, by student deep in the study of synthetic biology to write in an understanding manner, but sometimes one gets sloppy with the language for which most sincere apologies should suffice.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)
• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)
• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list represents a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in E. coli) by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into E. coli. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect E. coli cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

A synthetic oscillatory network of transcriptional regulators

2015-01-03T17:24:46Z

ValterB: New page: [http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators] Michael B. Elowitz & Stanislas Leibler Departments of ...

[http://http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]

Michael B. Elowitz & Stanislas Leibler
Departments of Molecular Biology and Physics, Princeton University, Princeton,
New Jersey 08544, USA

The passing of the millennia marked a profound paradigm shift in biological sciences. High-profile multinational projects were beginning to be organised but there was another meme on the move. A quieter, subtler movement was formed. It was not confined to research institutions – it spread to general population… To garages and basements, to inquisitive minds of people not strictly linked to biotechnology. Thirty, forty or perhaps even fifty years ago a storm of digital enthusiasm was raging through general population, making space for new mentality. In those bygone times the advance of electronic technology represented means to achievement, while the cause was always striving for something – for ideals and philosophies. Much like those bygone times were the first years of millennia and much like those bygone times are the passing years still. Except the technology has changed. Previously, digital was new, now new became biological. A tipping point was reached when enough analytical data accumulated to begin knitting it together. Much like a toddler breaks apart your carefully constructed plastic block houses, and does it again and again until some day he begins to stack the bricks together and sees that they stick. Not soon after he finds out that he’s quite good at it, that what he thinks will stick together actually sticks together. And so he begins to stack the foundations of what could someday become a house grander that everything you have ever shown him. They called the at the time emerging science Synthetic biology, an elusive field to grasp and confine still. It strives to create biological systems, the ones we observed in nature and the ones with functions we can only imagine. By biological systems we mean human-logical components of living organisms with distinct function like temperature sensors on our wrists, circadian clock that drives us to sleep or flagella on bacteria that enable them to swim. You will notice we will also use the term biological device throughout this chapter. A biological device is simply a less advanced biological system, but the terms are sometimes interchangeably used.

In the year 2000 a short paper was published in the Letters to nature. In it two physicists by education, Dr. Michael B. Elowitz and Dr. Stanislas Leiber, described the path to creation of an artificial oscillator based on known genetic components. They set off by making a simplified mathematical model of transcriptional regulation. The model, using a specific network of transcriptional regulators, predicted sustained oscillatory behaviour in some instances (figure 1b in the article), such that can be observed in many natural systems like circadian clock and similar. Their model accounts for mRNA and protein synthesis and degradation and the binding kinetics of operator proteins. Using numerical simulations, the authors have shown that such configurations do produce sustained oscillations while accounting for all known and predictable variables. Using numerical simulations they have also shown that previously reported stochastic variations can be responsible for the observed noise in gene expression networks. The short article helped initiate the emerging field of synthetic biology.

The inner working of an organism can be represented by networks of interacting molecules, carrying out a certain function in living cells. Such networks can be seen as cellular subsystems – independent mechanisms in intricate clockwork of a cell. They are created by scientists as human-readable representations of systems of biochemical reactions, constructed from data obtained in analytical experiments. Many, hundreds if not thousands and more of such discriminate networks, could possibly be combined to form an in silico model of a living organism. While the identification of such networks and the elucidation of their functions have been accomplished throughout the history of biological sciences, the underlying principles of their working are not so easy to uncover. Using modern synthetic approach to bioengineering, such understanding is not always required in order to replicate the work of nature.

== Introduction ==

In the above mentioned article the authors present the design and construction of an artificial biological system that implements a particular cellular function, existence of which was discovered by analysis of naturally occurring organisms. The system they chose to reverse engineer is a biological oscillator – a functional component of some living cells with a periodical output in a form of protein concentrations, not unlike a musical metronome or a clock. Many organisms use similar biological devices to adapt to periodic variations in environment such as the cycle of ebbs and tides or the day and night cycle. The most ground-breaking novelty described in the article is the fact that the artificial biological system was built using knowledge from a wide variety of biological sciences without intentionally, or knowingly, copying the nature’s design of a system with analogous function. This pioneering work is why this article ranks top in synthetic biology by the amount of citations. Such feat was accomplished by coupling a great deal of cleverness with accurate characterisation of a large amount of components of biological systems, such as promoters of RNA synthesis, binding proteins of various functional sites on DNA and similar. Only previously obtaining such high quality data could this task be accomplished and such data lies at the heart of at the time emerging science of synthetic biology. Its aim is to simplify work in the field of biological sciences and is known by experiments that aim to mimic or compliment nature’s work for the benefit of humanity. The vast majority of them use cutting-edge gene-manipulation technologies which produce transgenic organisms disfavoured by general public. A notable novelty of synthetic biology is the paradigm which favours simplicity – grab a basic DNA sequence, analyse what it does, change it slightly to ease manipulation with it, wrap it in a neat black box with all the properties easily accessible in an open database and call it a BioBrick or similar. These parts can be easily combined using simple yet clever molecular manipulation methods in order to create more complex molecular devices. Most advanced ideas involved in synthetic biology are regarded way ahead of our time; for more information reader should consult reports from iGEM (international Genetically Engineered Machine) competitions, which date back to 2003, or perhaps take a dive into the subculture of biohacking.

== Overview ==

The artificial oscillatory system, designed by the authors, will be inserted into host’s genomic material, also termed the chassis, and will essentially become just another system in the host organism. They chose Escherichia coli, the best studied organism as well as a simple prokaryote, as a biological chassis for their experiment. The reasoning behind this choice is solid – according to understanding at the time, E. coli regulatory systems should not interfere with the components of the artificial system, some of which are also, by nature, parts of regulatory systems. The design of the oscillator presumes no or benign interactions between its components and the components of host organism. For advanced readers the strain of the host E. coli should be mentioned – all experiments described in the article use strain MC4100. Its genotype is marked by substantial mutation that encompasses full lac operon; this removes the interference of additional binding sites for LacI repressor or induced or leaked expression of endogenous repressor protein. Other parts used in the design of the represillator are not found in E. coli genome and therefore should not harbour interactions with host matrix.

== Composition ==

At this point we should introduce the individual components of the oscillatory system termed ‘the represillator’ by the authors. Firstly, the name represillator suits the artificial system well – it is composed of three genes which code for proteins that act as repressors, expression of which is controlled in a cyclical way by promoters combined with operators they repress. The working of the circuit, told in a single sentence, may sound confusing for a general reader, much like some of the synthetic biology’s terminology used in the entire chapter. This chapter is written for semi-specialised public and many concepts are explained on site – despite this, the reader is encouraged to get a firm grasp on the meaning of terms before continuing. Great efforts were made, cross my heart, by student deep in the study of synthetic biology to write in an understanding manner, but sometimes one gets sloppy with the language for which most sincere apologies should suffice.

The represillator’s construction is evidently tripartite – it is formed by combining three similar parts, composition of which is as follows: first comes the promoter which is simply a sequence of DNA which promotes transcription of the sequence that immediately follows it. In and around it are the binding sites for the repressor proteins, also called operator sites – these are merely short sequences of DNA which assume such conformation that selectively binds repressor protein of certain type. When a repressor protein is bound to this part of DNA, the resulting complex inhibits the activity of a nearby promoter and greatly reduces transcription of the following genetic material. At the end of the promoter lie some sequences that are crucial for protein synthesis and are usually mentioned separately from the promoter – these are the ribosome binding site, more often than not shortened to an acronym RBS, and the starting codon. Immediately following the start codon is the coding region of the repressor protein. Information about amino acid sequence of the protein is stored in this comparatively large piece of DNA. It is followed by a stop codon and a transcription terminator, the first one interrupts the translation and the latter transcription of the gene at a specific spot; both are considered a mere bagatelle in most applications. The idea of cyclical repression is best conveyed with the help of figure 1a in the article. We have added a simplified representation of the aforementioned figure below, in the form of a list of components, for your convenience.

• promoter(λPR, repressed by λ cI)- LacI-lite (repressor)
• promoter(PLlacO1, repressed by LacI) - TetR-lite (repressor)
• promoter (PLtetO1, repressed by TetR) - λ cI-lite (repressor)

This list represents a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.

There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.

At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ PL promoter with lac and tet operator sequences termed PLlac01 and PLtet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in E. coli) by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.

At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into E. coli. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter PLtetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect E. coli cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.

== Results ==

As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at 30°C) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.

The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause other effects, such as substantial changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.

== Theoretical work ==

At the end of the article authors contemplate the theoretical basis behind the work, described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation in natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance; the authors ask whether construction of similar circuits that would retain this highly valued property is possible as a logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.

Negativna selekcija in spreminjanje striktnosti pri zvezni evoluciji s pomočjo fagov

2014-05-23T10:44:06Z

ValterB:

1. POVZETEK

Zvezna evolucija s pomočjo fagov (v nadaljevanju PACE) je metoda zvezne evolucije bioloških makromolekul. Zapis za te leži v genomu spremenjenih nitastih fagov, ki se propagirajo v obnavljajoči kulturi gostiteljskih E. coli. Lastnosti oziroma aktivnosti makromolekul so molekularno povezane s proliferativno sposobnostjo nosilnega nitastega faga. To odraža selekcijski pritisk za izboljšanje teh lastnosti makromolekule. V članku avtorji predstavijo dve izboljšavi njihove osnovne metode – uvedbo negativne selekcije in spreminjanje intenzitete selekcijskega pritiska. Njuno uporabnost prikažejo preko razvoja RNA polimeraze T7 s spremenjenim, in ne le razširjenim, prepoznavanjem promotorjev.

2. UVOD

Avtorji so zapis za fagni gen III, ki je ključen za njegovo infektivnost, prestavili na pomožni plazmid (ang. accessory plasmid, AP) gostiteljskih E. coli, v fagni genom pa vstavili zapis za RNA polimerazo T7. Gen III je v AP pod kontrolo promotorja PT3; neposredna posledica tega je selekcijski pritisk na RNA polimerazo T7 po prepoznavanju PT3. Tovrstni sistem so že uporabili v eksperimentu izpred treh let, vendar so naleteli na neuspeh – selekcijski pritisk je bil prevelik glede na velikost populacije oziroma njeno diverziteto, da bi omogočali razvoj ustreznih mutant, kar je povzročilo izumrtje faga. Le to so preprečili z uporabo PT7/T3 hibridega promotorja, ki je deloval kot evolucijski premostitveni kamen med PT7 in PT3.
Njihov namen je razviti splošen sistem za razvoj raznovrstnih makromolekul, uporaba premostitvenega kamna pa ni vedno enostavna ali sploh mogoča. S tem razlogom so v metodo uvedli nastavljivo intenziteto selekcijskega pritiska. Ker pa so želeli pripraviti makromolekule s spremenjenimi, in ne le razširjenimi, lastnostmi, so uvedli še metodo negativne selekcije proti prvotnim aktivnostim.

3. SPREMEMBA SELEKCIJSKEGA PRITISKA

V primerih visokega selekcijskega pritiska uspešna prilagoditev zahteva veliko populacijo in diverziteto, v nasprotnem primeru populacija nitastih fagov v mikrookolju izumre. V teh primerih bi radi znižali selekcijski pritisk in tako omogočili populaciji, da se na spremembo stopenjsko prilagodi. Avtorji so to dosegli z uvedbo dodatne kopije gena III na AP pod kontrolo inducibilnega promotorja. S tem lahko v določenem času inducirajo od razvijajoče molekule neodvisno izražanje gena III, kar efektivno zniža selekcijski pritisk. To omogoči nabiranje mutacij z, v teh pogojih majhnim, a znatnim vplivom na proliferativno sposobnost, ki pa s časom vodijo do razvoja molekul z dobrimi lastnostmi. V skrajnem primeru močne indukcije fagna populacija preide v stanje t. i. evolucijskega drsa (ang. evolutionary drift), ko nanje ne deluje znaten selekcijski pritisk.

4. NEGATIVNA SELEKCIJA

Za pripravo proteina s spremenjeno aktivnostjo so pripravili modifikacijo metode, pri kateri je prisoten negativni selekcijski pritisk proti prvotni aktivnosti. V idealnem primeru bi bila proliferativna sposobnost faga odvisna od razmerja med želeno in neželeno aktivnostjo. Prisotnost neželene funkcije mora tako zavreti proliferativno sposobnost faga, ozko grlo katere je v metodi PACE nivo pIII. Za to so avtorji uporabili antagonist pIII – mutirano obliko proteina pIII, t. i. N-C83 mutanto. Le ta se vgrajuje v fagne delce kjer povzroči zmanjšanje infektivnosti. Zapis za N-C83 gIII so vstavili v plazmid gostiteljskih celic pod kontrolo PT7, kar je povzročalo negativno selekcijo RNA polimeraz, ki ta promotor še prepoznavajo.

6. REZULTATI

Iz divjega tipa RNAP T7 so razvili mutanto RNA polimeraze T7 z višjo specifičnostjo za promotor PT3 napram PT7 od divjega tipa RNA polimeraze T7 za promotor PT7 napram PT3. Aktivnost razvitih mutant T7 na promotorju PT3 je bila višja od aktivnost divjega tipa RNAP T7 na lastnem promotorju PT7. Rezultata potrjujeta uspešno aplikacijo usmerjene molekularne evolucije z uporabo predstavljenih novosti.

Viri:
Negative selection and stringency modulation in phage-assisted continuous evolution (Jacob C. Carlson, Ahmed H. Badran, Drago A. Guggiana-Nilo & David R. Liu; Nature chemical biology 10, 216–222, 2014;

A system for the continuous directed evolution of biomolecules; Kevin M. Esvelt, Jacob C. Carlson and David R. Liu, Nature. 2011 April 28; 472(7344): 499–503.

Negativna selekcija in spreminjanje striktnosti pri zvezni evoluciji s pomočjo fagov.

2014-05-21T07:27:13Z

ValterB: New page: 1. POVZETEK Zvezna evolucija s pomočjo fagov (v nadaljevanju PACE) je metoda zvezne evolucije bioloških makromolekul. Zapis za te leži v genomu spremenjenih nitastih fagov, ki se propa...

1. POVZETEK

Zvezna evolucija s pomočjo fagov (v nadaljevanju PACE) je metoda zvezne evolucije bioloških makromolekul. Zapis za te leži v genomu spremenjenih nitastih fagov, ki se propagirajo v obnavljajoči kulturi gostiteljskih E. coli. Lastnosti oziroma aktivnosti makromolekul so molekularno povezane s proliferativno sposobnostjo nosilnega nitastega faga. To odraža selekcijski pritisk za izboljšanje teh lastnosti makromolekule. V članku avtorji predstavijo dve izboljšavi njihove osnovne metode – uvedbo negativne selekcije in spreminjanje intenzitete selekcijskega pritiska. Njuno uporabnost prikažejo preko razvoja RNA polimeraze T7 s spremenjenim, in ne le razširjenim, prepoznavanjem promotorjev.

2. UVOD

Avtorji so zapis za fagni gen III, ki je ključen za njegovo infektivnost, prestavili na pomožni plazmid (ang. accessory plasmid, AP) gostiteljskih E. coli, v fagni genom pa vstavili zapis za RNA polimerazo T7. Gen III je v AP pod kontrolo promotorja PT3; neposredna posledica tega je selekcijski pritisk na RNA polimerazo T7 po prepoznavanju PT3. Tovrstni sistem so že uporabili v eksperimentu izpred treh let, vendar so naleteli na neuspeh – selekcijski pritisk je bil prevelik glede na velikost populacije oziroma njeno diverziteto, da bi omogočali razvoj ustreznih mutant, kar je povzročilo izumrtje faga. Le to so preprečili z uporabo PT7/T3 hibridega promotorja, ki je deloval kot evolucijski premostitveni kamen med PT7 in PT3.
Njihov namen je razviti splošen sistem za razvoj raznovrstnih makromolekul, uporaba premostitvenega kamna pa ni vedno enostavna ali sploh mogoča. S tem razlogom so v metodo uvedli nastavljivo intenziteto selekcijskega pritiska. Ker pa so želeli pripraviti makromolekule s spremenjenimi, in ne le razširjenimi, lastnostmi, so uvedli še metodo negativne selekcije proti prvotnim aktivnostim.

3. SPREMEMBA SELEKCIJSKEGA PRITISKA

V primerih visokega selekcijskega pritiska uspešna prilagoditev zahteva veliko populacijo in diverziteto, v nasprotnem primeru populacija nitastih fagov v mikrookolju izumre. V teh primerih bi radi znižali selekcijski pritisk in tako omogočili populaciji, da se na spremembo stopenjsko prilagodi. Avtorji so to dosegli z uvedbo dodatne kopije gena III na AP pod kontrolo inducibilnega promotorja. S tem lahko v določenem času inducirajo od razvijajoče molekule neodvisno izražanje gena III, kar efektivno zniža selekcijski pritisk. To omogoči nabiranje mutacij z, v teh pogojih majhnim, a znatnim vplivom na proliferativno sposobnost, ki pa s časom vodijo do razvoja molekul z dobrimi lastnostmi. V skrajnem primeru močne indukcije fagna populacija preide v stanje t. i. evolucijskega drsa (ang. evolutionary drift), ko nanje ne deluje znaten selekcijski pritisk.

4. NEGATIVNA SELEKCIJA

Za pripravo proteina s spremenjeno aktivnostjo so pripravili modifikacijo metode, pri kateri je prisoten negativni selekcijski pritisk proti prvotni aktivnosti. V idealnem primeru bi bila proliferativna sposobnost faga odvisna od razmerja med želeno in neželeno aktivnostjo. Prisotnost neželene funkcije mora tako zavreti proliferativno sposobnost faga, ozko grlo katere je v metodi PACE nivo pIII. Za to so avtorji uporabili antagonist pIII – mutirano obliko proteina pIII, t. i. N-C83 mutanto. Le ta se vgrajuje v fagne delce kjer povzroči zmanjšanje infektivnosti. Zapis za N-C83 gIII so vstavili v plazmid gostiteljskih celic pod kontrolo PT7, kar je povzročalo negativno selekcijo RNA polimeraz, ki ta promotor še prepoznavajo.

6. REZULTATI

Iz divjega tipa RNAP T7 so razvili mutanto RNA polimeraze T7 z višjo specifičnostjo za PT3 napram promotorju PT7 od w. t. RNAP T7 za PT7 napram PT3. Aktivnost razvitih mutant T7 na promotorju PT3 je bila višja od aktivnost w. t. RNAP T7 na PT7. Rezultata potrjujeta uspešno aplikacijo usmerjene molekularne evolucije z uporabo predstavljenih novosti.

Viri:
Negative selection and stringency modulation in phage-assisted continuous evolution (Jacob C. Carlson, Ahmed H. Badran, Drago A. Guggiana-Nilo & David R. Liu; Nature chemical biology 10, 216–222, 2014;

A system for the continuous directed evolution of biomolecules; Kevin M. Esvelt, Jacob C. Carlson and David R. Liu, Nature. 2011 April 28; 472(7344): 499–503.

MBT seminarji 2014

2014-05-21T07:26:37Z

ValterB:

'''Seznam seminarjev iz Molekularne biotehnologije v študijskem letu 2013/14'''

V študijskem letu 13/14 izvajamo predmet Molekularna biotehnologija (in s tem tudi seminarje) prvič.
Tabela za razpored po tednih bo objavljena v spletni učilnici, vanjo pa se vpišite tudi za kratke predstavitve novic (5 min). Na tej strani bo samo seznam odobrenih člankov za seminar in povezave do člankov in do povzetkov, ki jih morate objaviti najkasneje tri dni pred predstavitvijo (petek).

Način vnosa:

# The importance of ''Arabidopsis'' glutathione peroxidase 8 for protecting ''Arabidopsis'' plant and ''E. coli'' cells against oxidative stress (A. Gaber; GM Crops & Food 5(1), 2014; http://dx.doi.org/10.4161/gmcr.26979) Pomen glutation peroksidaze 8 iz repnjakovca za zaščito rastline ''Arabidopsis thaliana'' in bakterije ''Escherichia coli'' pred oksidativnim stresom. Janez Novak, 15. marca 2014
(slovenski naslov povežete z novo stranjo, na kateri bo povzetek)

Naslovi odobrenih člankov:

# A plant factory for moth pheromone production (B-J. Ding ''et al''.; Nature Communications 5, 3353, 2014; http://www.nature.com/ncomms/2014/140225/ncomms4353/full/ncomms4353.html) [[Proizvodnja feremonov vešče v rastlinah]]. Filip Kolenc, 24. marca 2014
# Introduction of the rd29A:AtDREB2A CA gene into soybean (Glycine max L. Merril) and its molecular characterization in leaves and roots during dehydration (C. Engels ''et al''.; Genetics and Molecular Biology 36(4): 556–565, 2013; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873188/) [[Vstavitev gena rd29A:AtDREB2A CA v sojo in njegova molekulska karakterizacija v listih in koreninah med dehidracijo]]. Aleksander Krajnc, 24. marca 2014
# Enantioselective lactic acid production by an Enterococcus faecium strain showing potential in agro-industrial waste bioconversion: Physiological and proteomic studies (A. Pessione ''et al''.; Journal of Biotechnology 173, 31–40, 2014; http://dx.doi.org/10.1016/j.jbiotec.2014.01.014) [[Produkcija optično čiste mlečne kisline v sevu enterococcus faecium kaže potencial v biopretvorbi odpadkov kmetijske industrije: fiziološka in proteomska študija]]. Žan Železnik, 31. marca
# Isolation and characterization of formaldehyde-degrading fungi and its formaldehyde metabolism (D. Yu ''et al''.; Environmental Science and Pollution Research 2014 - v tisku; http://dx.doi.org/10.1007/s11356-014-2543-2) [[Glive, sposobne razgradnje formaldehida: izolacija, karakterizacija in njihov metabolizem formaldehida.]] Sara Sajko, 31. marca
# Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface (S. M. Lewis et al.; Nature Biotechnology 32, 191–198, 2014; http://www.nature.com/nbt/journal/v32/n2/full/nbt.2797.html) [[Priprava bispecifičnih IgG protiteles s pomočjo ustvarjanja strukturno baziranega ortogonalnega Fab vmesnika.]] Vito Frančič, 7. aprila
# Generation of protective immune response against anthrax by oral immunization with protective antigen plant-based vaccine (J. Gorantala, ''et al''; Journal of Biotechnology, 176, 2014, str. 1-10.; http://www.sciencedirect.com/science/article/pii/S0168165614000571) - [[Pridobitev zaščitnega imunskega odziva proti antraksu preko oralne imunizacije z zaščitnim antigenom kot cepivom, pridobljenim na osnovi rastlin]]. Sabina Kolar, 7. aprila
# Development of influenza H7N9 virus like particle (VLP) vaccine: Homologous A/Anhui/1/2013 (H7N9) protection and heterologous A/chicken/Jalisco/CPA1/2012 (H7N3) cross-protection in vaccinated mice challenged with H7N9 virus (G. E. Smith ''et al''.; Vaccine 31, 4305-4313, 2013; http://www.sciencedirect.com/science/article/pii/S0264410X13009870). [[Razvoj cepiva za virus gripe H7N9 na osnovi virusu podobnih delcev]]. Ana Dolinar, 14. aprila
# Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy (M. Themeli ''et al.''; Nature Biotechnology 31, 928–933, 2013; http://www.nature.com/nbt/journal/v31/n10/full/nbt.2678.html). [[Iz induciranih pluripotentnih izvornih celic pripravljeni človeški limfociti T za terapijo raka]]. Urban Bezeljak, 14. aprila
# Engineering ''Escherichia coli'' for selective geraniol production with minimized endogenous dehydrogenation (J. Zhou; Journal of Biotechnology 169, 2014; http://www.sciencedirect.com/science/article/pii/S016816561300494X) [[Inženiring Escherichie coli za selektivno produkcijo geraniola z minimalno endogeno dehidrogenacijo]]. Maja Remškar, 5. maja
# Identifying producers of antibacterial compounds by screening for antibiotic resistance. (M. N. Thaker et al.; Nature Biotechnology 31, 922-927; 2013). [[Identifikacija proizvajalcev antibakterijskih spojin z iskanjem odpornosti proti antibiotikom]]. Špela Podjed, 5. maja
# Consolidated conversion of protein waste into biofuels and ammonia using Bacillus subtilis (K-Y. Choi ''et al''.; Metabolic Engineering 2014 - v tisku; http://dx.doi.org/10.1016/j.ymben.2014.02.007). [[Pretvorba proteinskih odpadkov v biogoriva in amonijak z bakterijo B. subtilis]] Elmina Handanović, 12. maja 2014
# Transcriptional comparison of the filamentous fungus Neurospora crassa growing on three major monosaccharides D-glucose, D-xylose and L-arabinose (J. Li ''et al''.; Biotechnology for Biofuels 7:31, 2014; http://www.biotechnologyforbiofuels.com/content/7/1/31/abstract). [[Primerjava transkriptoma filamentoznih gliv Neurospora crassa pri rasti na treh različnih vrstah monosaharidov: D-glukoze, D-ksiloze in L-arabinoze]] Luka Bevc, 12. maja
# Influence of valine and other amino acids on total diacetyl and 2,3-pentanedione levels during fermentation of brewer’s wort. (K. Krogerus, et al., Microbiol Biotechnol. 2013 Aug; http://link.springer.com/article/10.1007%2Fs00253-013-4955-1). [[Vpliv valina in drugih aminokislin na vsebnost diacetila in 2,3-pentadiona v pivini]] Jernej Mustar, 19. maja
# Xylanase and cellulase systems of Clostridium sp.: An insight on molecular approaches for strain improvement (L. Thomas ''et al''.; Bioresource Technology 2014 - v tisku; http://dx.doi.org/10.1016/j.biortech.2014.01.140)[[Ksilanazni ter celulosomski sistemi klostridij:vpogled v molekularni pristoti za izboljšavo sevov]] Luka Grm, 19. maja
# M Cell-Targeting Ligand and Consensus Dengue Virus Envelope Protein Domain III Fusion Protein Production in Transgenic Rice Calli (Tae-Geum K.''et al''.; Molecular Biotechnology 54, 880-887, 2013; http://link.springer.com/article/10.1007%2Fs12033-012-9637-1 ) Veronika Jarc, 26. maja
# Negative selection and stringency modulation in phage-assisted continuous evolution (Jacob C. Carlson, Ahmed H. Badran, Drago A. Guggiana-Nilo & David R. Liu; Nature chemical biology 10, 216–222, 2014; http://www.nature.com/nchembio/journal/v10/n3/full/nchembio.1453.html) [[Negativna selekcija in spreminjanje striktnosti pri zvezni evoluciji s pomočjo fagov.]] Valter Bergant, 26. maja

Negativna selekcija in spreminjanje striktnosti pri zvezni evoluciji s pomočjo fagov

2014-05-21T07:25:47Z

1. POVZETEK

Zvezna evolucija s pomočjo fagov (v nadaljevanju PACE) je metoda zvezne evolucije bioloških makromolekul. Zapis za te leži v genomu spremenjenih nitastih fagov, ki se propagirajo v obnavljajoči kulturi gostiteljskih E. coli. Lastnosti oziroma aktivnosti makromolekul so molekularno povezane s proliferativno sposobnostjo nosilnega nitastega faga. To odraža selekcijski pritisk za izboljšanje teh lastnosti makromolekule. V članku avtorji predstavijo dve izboljšavi njihove osnovne metode – uvedbo negativne selekcije in spreminjanje intenzitete selekcijskega pritiska. Njuno uporabnost prikažejo preko razvoja RNA polimeraze T7 s spremenjenim, in ne le razširjenim, prepoznavanjem promotorjev.

2. UVOD

Avtorji so zapis za fagni gen III, ki je ključen za njegovo infektivnost, prestavili na pomožni plazmid (ang. accessory plasmid, AP) gostiteljskih E. coli, v fagni genom pa vstavili zapis za RNA polimerazo T7. Gen III je v AP pod kontrolo promotorja PT3; neposredna posledica tega je selekcijski pritisk na RNA polimerazo T7 po prepoznavanju PT3. Tovrstni sistem so že uporabili v eksperimentu izpred treh let, vendar so naleteli na neuspeh – selekcijski pritisk je bil prevelik glede na velikost populacije oziroma njeno diverziteto, da bi omogočali razvoj ustreznih mutant, kar je povzročilo izumrtje faga. Le to so preprečili z uporabo PT7/T3 hibridega promotorja, ki je deloval kot evolucijski premostitveni kamen med PT7 in PT3.
Njihov namen je razviti splošen sistem za razvoj raznovrstnih makromolekul, uporaba premostitvenega kamna pa ni vedno enostavna ali sploh mogoča. S tem razlogom so v metodo uvedli nastavljivo intenziteto selekcijskega pritiska. Ker pa so želeli pripraviti makromolekule s spremenjenimi, in ne le razširjenimi, lastnostmi, so uvedli še metodo negativne selekcije proti prvotnim aktivnostim.

3. SPREMEMBA SELEKCIJSKEGA PRITISKA

V primerih visokega selekcijskega pritiska uspešna prilagoditev zahteva veliko populacijo in diverziteto, v nasprotnem primeru populacija nitastih fagov v mikrookolju izumre. V teh primerih bi radi znižali selekcijski pritisk in tako omogočili populaciji, da se na spremembo stopenjsko prilagodi. Avtorji so to dosegli z uvedbo dodatne kopije gena III na AP pod kontrolo inducibilnega promotorja. S tem lahko v določenem času inducirajo od razvijajoče molekule neodvisno izražanje gena III, kar efektivno zniža selekcijski pritisk. To omogoči nabiranje mutacij z, v teh pogojih majhnim, a znatnim vplivom na proliferativno sposobnost, ki pa s časom vodijo do razvoja molekul z dobrimi lastnostmi. V skrajnem primeru močne indukcije fagna populacija preide v stanje t. i. evolucijskega drsa (ang. evolutionary drift), ko nanje ne deluje znaten selekcijski pritisk.

4. NEGATIVNA SELEKCIJA

Za pripravo proteina s spremenjeno aktivnostjo so pripravili modifikacijo metode, pri kateri je prisoten negativni selekcijski pritisk proti prvotni aktivnosti. V idealnem primeru bi bila proliferativna sposobnost faga odvisna od razmerja med želeno in neželeno aktivnostjo. Prisotnost neželene funkcije mora tako zavreti proliferativno sposobnost faga, ozko grlo katere je v metodi PACE nivo pIII. Za to so avtorji uporabili antagonist pIII – mutirano obliko proteina pIII, t. i. N-C83 mutanto. Le ta se vgrajuje v fagne delce kjer povzroči zmanjšanje infektivnosti. Zapis za N-C83 gIII so vstavili v plazmid gostiteljskih celic pod kontrolo PT7, kar je povzročalo negativno selekcijo RNA polimeraz, ki ta promotor še prepoznavajo.

6. REZULTATI

Iz divjega tipa RNAP T7 so razvili mutanto RNA polimeraze T7 z višjo specifičnostjo za PT3 napram promotorju PT7 od w. t. RNAP T7 za PT7 napram PT3. Aktivnost razvitih mutant T7 na promotorju PT3 je bila višja od aktivnost w. t. RNAP T7 na PT7. Rezultata potrjujeta uspešno aplikacijo usmerjene molekularne evolucije z uporabo predstavljenih novosti.

Viri:
Negative selection and stringency modulation in phage-assisted continuous evolution (Jacob C. Carlson, Ahmed H. Badran, Drago A. Guggiana-Nilo & David R. Liu; Nature chemical biology 10, 216–222, 2014;

A system for the continuous directed evolution of biomolecules; Kevin M. Esvelt, Jacob C. Carlson and David R. Liu, Nature. 2011 April 28; 472(7344): 499–503.

MBT seminarji 2014

2014-03-04T17:29:33Z

ValterB:

ScFv phage display

2013-11-01T18:22:52Z

ValterB: New page: === Seminarska naloga – analiza članka An efficient method for isolating antibody fragments against small peptides by antibody phage display === Zhi Duan and Henrik Siegumfeldt, Combina...

=== Seminarska naloga – analiza članka An efficient method for isolating antibody fragments against small peptides by antibody phage display ===
Zhi Duan and Henrik Siegumfeldt, Combinatorial chemistry & high throughput screening, 2010, 13, 818-828
----

=== POVZETEK ===
Cilj članka je bila priprava človeških enoverižnih variabilnih fragmentov (HuscFv) proti trem kratkim peptidom (dolžine dvakrat 15 in enkrat 14 aminokislin) z uporabo predstavitve na nitastih fagih. Peptidi so del αs1-kazeina in vsebujejo prepoznavna mesta za različne encime, ki sodelujejo pri zorenju sira; namen pripravle protiteles je raziskovanje poteka tega pojava. Avtorja sta predstavila težave, s katerimi sta se soočila pri pripravi protiteles – pri immobilizaciji antigenov in pri zmanjševanju nespecifične vezave fagnih delcev. Opisala sta postopke dela, prilagojene za pripravo protiteles proti kratkim polipeptidom in iz knjižnic Thomlinson I in J izolirala visoko specifična protitelesa proti vsem trem antigenom. Ovrednotila sta tudi njihovo vezavo na izvorni in sorodni nativni kazein.

=== Določitev kapacitete vezave trdnih nosilcev ===
V začetnem delu postopka obogatitve knjižnice je potrebno na trdni nosilec učinkovite vezati ustrezne antigene. Avtorja sta ob uporabi kratkih peptidov naletela na več ovir. Majhna velikost polipeptidov zmanjša zmožnost vezave na pasivne adsorbente, lahko se pojavijo tudi težave pri kovalentnih vezavah. Njuna tehnika določitve zasedenosti nosilca (vdolbina na mikrotitrski plošči) z antigenom je temeljila na vezavi antigena na nosilec po standardnem postopku. Za tem je sledila vezava pomožnega faga KM13 na isti nosilec. Ti naj bi zasedli vsa preostala prosta mesta, množina vezanega pomožnega faga pa naj bi bila merilo za zasedenost nosilca z antigenom. Kapaciteto vezave sta določila trem trdnim nosilcem z različnimi načini vezave antigena:

Immo-Strep*: imobilizacija preko vezave biotiniliranih antigenov na imobiliziran streptavidin (pozitivna kontrola – biotin, negativna kontrola - nič)
Immo-Amino*: kovalentna imobilizacija preko aminskih (in drugih nukleofilnih) skupin (pozitivna kontrola – BSA in kazein (CN), negativna kontrola - nič)
Maxisorp: pasivna adsorpcija (pozitivna kontrola – BSA in CN, negativna kontrola - nič)

* Osnova aktivne površine je Polysorp (polistiren), zaradi česar so možne tudi nespecifične hidrofobne interakcije

Množino vezanega KM13 so določili po inkubacija s HRP/anti-M13 protitelesi in kromogeni encimski reakcij spektrofotometrično. Izkazalo se je, da je zasedenost nosilca zaradi vezave antigena na Immo-Amino in Maxisorp pod mejo zaznave – rezultati za vse tri antigene se statistično niso razlikovali od negativne kontrole. Nizko zmožnost vezave antigenov na ta dva nosilca so potrdili tudi na koncu z novo pripravljenimi protitelesi proti vsem trem kratkim peptidom – količina vezanih peptidov je bila pod mejo detekcije.

Za razliko od prejšnjih dveh pa je bila zasedenost površine nosilca zaradi vezave antigena v primeru uporabe Immo-Strep enaka pozitivni kontroli (presežni biotin). Za pripravo vseh protiteles, omenjenih v članku, so uporabili ta nosilec.

Rezultati vezave so v splošnem odvisi od antigena. V diskusiji avtorja omenita, da so drugi raziskovalci uspešno uporabili prej omenjene metode za vezavo podobnih antigenov.

=== Poskus priprave protiteles proti kratkemu peptidu F2 ===
scFv-ji so v knjižnicah Thomlinson I in J predstavljeni kot fuzijski proteini s proteinom pIII. Med fagni protein in scFv je bil vstavljen še c-myc tag, ki vsebuje prepoznavno mesto za tripsin. Aminokislinsko zaporedje proteina pIII pomožnega faga KM13 je bilo spremenjeno tako, da prav tako vsebuje prepoznavno mesto za to proteazo. Elucija vezanih scFv-fagov s tripsinom (TE) tako sprosti fagne delce z zapisi za scFv-je, ki z nosilcem interagirajo preko protiteles. Po inkubaciji s tem encimom pomožni fag ni več zmožen infekcije. Tovrstno elucijo so uporabili v prvotnem postopku selekcije protiteles proti peptidu F2.

Po štirih krogih selekcije in ovrednotenju specifičnosti naključnih 2x96 (knjižnici I in J) scFv-jev nista dobila protiteles, ki bi specifično vezali antigen. Dobila sta več kot sto pozitivnih interakcij z nosilcem , tarče preostalih protiteles pa so neznane.

=== Ovrednotenje dveh drugih metod elucije ===
Zaradi negativnih rezultatov z uporabo elucije s tripsinom sta selekcijo na F2 izvedla še z dvema spodaj opisanima metodama elucije:

kompetitivna elucija (CE): elucija s presežnim antigenom

elucija z naknadno površinsko adsorpcijo (SA): elucija s trietilaminom in naknadna vezava eluata na prost nosilec. Nanj se vežejo protitelesa, ki interagirajo z nosilcem, tista,
ki vežejo antigen pa se neovirano sperejo.

Po štirih krogih selekcije na vsak antigen so avtorji testirali specifičnost naključnih 96 klonov . Z uporabo metode kompetitivne elucije sta dobila boljše rezultate. Po analogni metodi obogatitve knjižnic sta avtorja pridobila več kot 120 klonov (ne nujno unikatnih), ki vežejo antigen F2. Tudi elucija z naknadno površinsko adsorpcijo je dala dobre rezultate z uporabo knjižnice Thomlinson J, z uporabo knjižnice I pa zaradi neznanih razlogov rezultate, podobne uporabi elucije s tripsinom. Zaradi tega sta avtorja za pripravo protiteles proti F1 in F3 uporabila le knjižnico J.

=== Rezultati ===
Metoda selekcije z uporabo CE je dala pozitivne rezultate za vse tri antigene, proti peptidu F1 je bilo pripravljeno in ovrednoteno eno, proti peptidu F2 dve, proti peptidu F3 pa pet edinstvenih protiteles. Vsem protitelesom proti antigenom je bila tudi določena specifičnost s testom vezave več nespecifičnih tarč (BSA, Gelatin, ostali antigeni) in nekaj pozitivnih tarč (lasten antigen, αs-kazein, soroden kazein). Pripravljeni sta bili tudi dve protitelesi proti Polysorp-u, osnovi trdnega nosilca. Izmed pripravljenih protiteles so tri vsebovala le lahko verigo, kar jih uvršča med enodomenska protitelesa, bolj znana kot nanotelesca.

Seminarji TehDNA

2013-10-15T10:43:48Z

ValterB:

Seminarje iz Tehnologije DNA bo v študijskem letu 2013/14 vodila asist. dr. Helena Čelešnik.

Seznam tem za seminarje:

# Mutageneza (16.10.) (povzetek in porocilo lahko oddate v pon., 14.10), 3 seminarji: 1. Urban Bezeljak (CRISPR/Cas9) 2. Uroš Stupar (ZFN nukleaze) 3. Helena Vajović (tarčna mutageneza)
# Izražanje na površini (23.10.), 3 seminarji: 1. Mitja Crček (Ribosome display), 2. Klara Tereza Novoselc (Phage displey) 3. Živa Marsetič
# Dvohibridni sistemi (30.10.), 3 seminarji: 1. Katja Kovačič 2. Barbara Žužek 3. Bernarda Majc
# Mutageneza, izražanje na površini ali dvohibridni sistemi (6.11.), 3 seminarji: 1. Valter Bergant (scFv phage display), 2. Ana Kapraljević, 3. Tjaša Blatnik
# GSO v agronomiji (13.11.), 3 seminarji: 1. Niki Bursič, 2. Petra Malavašič, 3. Jernej Mustar
# Transgenske živali (27.11.), 3 seminarji: 1. Andrea Grof, 2. Eva Lucija Kozak, 3. Špela Pohleven
# Izvorne celice (4.12.), 4 seminarji: 1. Sara Primec, 2. Alja Zottel, 3. Tjaša Goričan, 4. Rok Štemberger
# DNA-diagnostika (11.12.), 4 seminarji: 1. Tina Gregorič , 2. Eva Knapič, 3. Veronika Jarc, 4. Jana Verbančič
# Forenzika, arheologija, sistematika (18.12.), 3 seminarji: 1. Matja Zalar, 2. Andreja Bratovš, 3. Maja Remškar
# Mikromreže, genomike (8.1.), 3 seminarji: 1. Andrej Vrankar, 2. Filip Kolenc 3. Nastja Štemberger
# Gensko zdravljenje s. lat. (15.1.), 3 seminarji: 1. Ana Dolinar 2. Staša Komljenovič, 3. Katarina Uršič

'''IZBIRANJE ČLANKOV ZA SEMINARJE:
Ni nujno, da je metoda, ki jo želimo predstaviti, sama tematika izbranega članka. Zaželeno je, da članek obravnava neko biološko temo, pri raziskovanju le-te pa avtorji uporabljajo metodo, ki jo želimo predstaviti.'''

'''POVZETKI ZA SEMINARJE 14.10.2013'''

1. Urban Bezeljak ([[CRISPR/Cas9]])

2. Uroš Stupar (ZFN nukleaze)

3. Helena Vajović ([[tarčna mutageneza]])

Seminarji TehDNA

2013-10-08T16:20:14Z

ValterB:

Seminarje iz Tehnologije DNA bo v študijskem letu 2013/14 vodila asist. dr. Helena Čelešnik.

Seznam tem za seminarje:

# Mutageneza (16.10.), 3 seminarji:
# Izražanje na površini (23.10.), 3 seminarji:
# Dvohibridni sistemi (30.10.), 3 seminarji:
# Mutageneza, izražanje na površini ali dvohibridni sistemi (6.11.), 3 seminarji: 1. Valter Bergant
# GSO v agronomiji (13.11.), 3 seminarji: 1. Niki Bursič, 2. Petra Malavašič, 3. Jernej Mustar
# Transgenske živali (27.11.), 3 seminarji: 1. Andrea Grof, 2. Eva Lucija Kozak, 3. Špela Pohleven
# Izvorne celice (4.12.), 4 seminarji: 1. Sara Primec, 2. Alja Zottel, 3. Tjaša Goričan, 4. Rok Štemberger
# DNA-diagnostika (11.12.), 4 seminarji: 1. Tina Gregorič , 2. Eva Knapič, 3. Veronika Jarc, 4. Jana Verbančič
# Forenzika, arheologija, sistematika (18.12.), 3 seminarji: 1. Matja Zalar, 2. Andreja Bratovš, 3. Maja Remškar
# Mikromreže, genomike (8.1.), 3 seminarji: 1. Andrej Vrankar
# Gensko zdravljenje s. lat. (15.1.), 3 seminarji: 1. Ana Dolinar

Luciferaza

2010-11-13T08:59:14Z

ValterB: /* Luciferaza kresničk */

== Luciferaza ==

Luciferaza[http://en.wikipedia.org/wiki/File:Firefly_Luciferase_Crystal_Structure.rsh.png] je ime za vrsto proteinskih encimov, ki sodelujejo pri reakcijah bioluminiscence v različnih živih bitjih. Običajno se to ime uporablja za encim v kresničkah (''Photinus Pyralis'', EC|1.13.12.7). Ime je izpeljanka iz latinskih besed lucem ferre, kar pomeni prinašalec luči.

Različne variacije tega encima se nahajajo v družini kresničk in hroščev, v gobah (kot je ''Omphalotus Olearius''[http://en.wikipedia.org/wiki/Omphalotus_olearius]) in veliko morskih organizmih (meduze, sipe, globokomorske ribe, mikroorganizmi). Razvili so se neodvisno in se razlikujejo po sestavi, substratih in valovni dolžini emitirane svetlobe. Prvo reakcijo luciferin-luciferaza je izvedel francoz Horace Raphael Dubois leta 1885. Iz svetilnih organov hroščev iz roda ''Pyrophorus'' je posebej ekstrahiral encim v hladnem, in substrat v vročem, kjer je encim uničen. Ta dva ekstrakta je nato zmešal, in opazoval svetlobo, ki se je pri tem sproščala. Encim in pripadajoč substrat je tudi poimenoval.
Gen za ta encim so že vgradili v genski zapis raznih višjih organizmov, kot sta miš, zajec in krompir.
Encim se da pripeti na določene molekule (na primer protitelesa), in z emitirano svetlobo spremljati njihov položaj.
Uporablja se tudi za merjenje koncentracije ATP v vzorcih, na podoben način pa se uporablja tudi za detekcijo sledov krvi v forenziki.

== Luciferaza kresničk ==

Pri reakciji bioluminiscence kot kofaktorja nastopata [[ATP]] in magnezijev ion, substrat pa je [[luciferin]]. Optimalna temperatura za delovanje encima iz kresničk je 23-25°C, optimalen pH 7.8, molska masa encima pa med 60000 in 62000 Daltoni. Pri bakterijah molska masa znaša okoli 80000 Daltonov, optimalen pH pa je večinoma pod 7.
Encim v prisotnosti ATP in magnezijevega iona katalizira oksidacijo luciferina do hidroperoksidnega intermediata, ki nato ciklizira in pri razpadu odda svetlobo. Reakcija ima zelo dober izkoristek, saj je skoraj vsa energija sproščena v obliki svetlobe.
Že zelo majhna sprememba proteinskega encima luciferaze (en aminokislinski ostanek) se odraža na spremembi valovne dolžine emitirane svetlobe, zato različne vrste kresničk, ki imajo majhne variacije v genskem zapisu za ta protein, oddajajo svetlobo različnih valovnih dolžin (552-582nm, med rumeno in zeleno barvo).

== Viri ==

Worthington Biochemical Corporation - http://www.worthington-biochem.com/lu/default.html

Wapedia - http://wapedia.mobi/sl/Bioluminiscenca

Britannica - http://www.britannica.com/EBchecked/topic/350599/luciferase

Bioluminescence - Chemical Principles and Methods, Osamu Shimomura, World scientific Publishing - 2006

[[Category:LEX]]

Luciferaza

2010-11-06T15:51:23Z

ValterB: /* Luciferaza */

== Luciferaza ==

Luciferaza[http://en.wikipedia.org/wiki/File:Firefly_Luciferase_Crystal_Structure.rsh.png] je ime za vrsto proteinskih encimov, ki sodelujejo pri reakcijah bioluminiscence v različnih živih bitjih. Običajno se to ime uporablja za encim v kresničkah (''Photinus Pyralis'', EC|1.13.12.7). Ime je izpeljanka iz latinskih besed lucem ferre, kar pomeni prinašalec luči.

Različne variacije tega encima se nahajajo v družini kresničk in hroščev, v gobah (kot je ''Omphalotus Olearius''[http://en.wikipedia.org/wiki/Omphalotus_olearius]) in veliko morskih organizmih (meduze, sipe, globokomorske ribe, mikroorganizmi). Razvili so se neodvisno in se razlikujejo po sestavi, substratih in valovni dolžini emitirane svetlobe. Prvo reakcijo luciferin-luciferaza je izvedel francoz Horace Raphael Dubois leta 1885. Iz svetilnih organov hroščev iz roda ''Pyrophorus'' je posebej ekstrahiral encim v hladnem, in substrat v vročem, kjer je encim uničen. Ta dva ekstrakta je nato zmešal, in opazoval svetlobo, ki se je pri tem sproščala. Encim in pripadajoč substrat je tudi poimenoval.
Gen za ta encim so že vgradili v genski zapis raznih višjih organizmov, kot sta miš, zajec in krompir.
Encim se da pripeti na določene molekule (na primer protitelesa), in z emitirano svetlobo spremljati njihov položaj.
Uporablja se tudi za merjenje koncentracije ATP v vzorcih, na podoben način pa se uporablja tudi za detekcijo sledov krvi v forenziki.

== Luciferaza kresničk ==

Pri reakciji bioluminiscence kot kofaktorja nastopata [[ATP]] in magnezijev ion, substrat pa je [[luciferin]]. Optimalna temperatura za delovanje encima iz kresničk je 23-25°C, optimalen pH 7.8, molska masa encima pa med 60000 in 62000 Daltoni. Pri bakterijah molska masa znaša okoli 80000 Daltonov, optimalen pH pa je večinoma pod 7.
Encim v prisotnosti ATP in magnezijevega iona katalizira oksidacijo luciferina do hidroperoksidnega intermediata, ki nato ciklizira in pri dekarboksilaciji odda svetlobo. Reakcija ima zelo dober izkoristek, saj je skoraj vsa energija sproščena v obliki svetlobe.
Že zelo majhna sprememba proteinskega encima luciferaze (en aminokislinski ostanek) se odraža na spremembi valovne dolžine emitirane svetlobe, zato različne vrste kresničk, ki imajo majhne variacije v genskem zapisu za ta protein, oddajajo svetlobo različnih valovnih dolžin (552-582nm, med rumeno in zeleno barvo).

== Viri ==

Worthington Biochemical Corporation - http://www.worthington-biochem.com/lu/default.html

Wapedia - http://wapedia.mobi/sl/Bioluminiscenca

Britannica - http://www.britannica.com/EBchecked/topic/350599/luciferase

Bioluminescence - Chemical Principles and Methods, Osamu Shimomura, World scientific Publishing - 2006

[[Category:LEX]]

Luciferaza

2010-11-05T20:02:07Z

ValterB: /* Luciferaza kresničk */

== Luciferaza ==

Luciferaza[http://en.wikipedia.org/wiki/File:Firefly_Luciferase_Crystal_Structure.rsh.png] je ime za vrsto proteinskih encimov, ki sodelujejo pri reakcijah bioluminiscence v različnih živih bitjih. Običajno se to ime uporablja za encim v kresničkah (''Photinus Pyralis''). Ime je izpeljanka iz latinskih besed lucem ferre, kar pomeni prinašalec luči.

Različne variacije tega encima se nahajajo v družini kresničk in hroščev, v gobah (kot je ''Omphalotus Olearius''[http://en.wikipedia.org/wiki/Omphalotus_olearius]) in veliko morskih organizmih (meduze, sipe, globokomorske ribe, mikroorganizmi). Razvili so se neodvisno in se razlikujejo po sestavi, substratih in valovni dolžini emitirane svetlobe. Prvo reakcijo luciferin-luciferaza je izvedel francoz Horace Raphael Dubois leta 1885. Iz svetilnih organov hroščev iz roda ''Pyrophorus'' je posebej ekstrahiral encim v hladnem, in substrat v vročem, kjer je encim uničen. Ta dva ekstrakta je nato zmešal, in opazoval svetlobo, ki se je pri tem sproščala. Encim in pripadajoč substrat je tudi poimenoval.
Gen za ta encim so že vgradili v genski zapis raznih višjih organizmov, kot sta miš, zajec in krompir.
Encim se da pripeti na določene molekule (na primer protitelesa), in z emitirano svetlobo spremljati njihov položaj.
Uporablja se tudi za merjenje koncentracije ATP v vzorcih, na podoben način pa se uporablja tudi za detekcijo sledov krvi v forenziki.

== Luciferaza kresničk ==

Pri reakciji bioluminiscence kot kofaktorja nastopata [[ATP]] in magnezijev ion, substrat pa je [[luciferin]]. Optimalna temperatura za delovanje encima iz kresničk je 23-25°C, optimalen pH 7.8, molska masa encima pa med 60000 in 62000 Daltoni. Pri bakterijah molska masa znaša okoli 80000 Daltonov, optimalen pH pa je večinoma pod 7.
Encim v prisotnosti ATP in magnezijevega iona katalizira oksidacijo luciferina do hidroperoksidnega intermediata, ki nato ciklizira in pri dekarboksilaciji odda svetlobo. Reakcija ima zelo dober izkoristek, saj je skoraj vsa energija sproščena v obliki svetlobe.
Že zelo majhna sprememba proteinskega encima luciferaze (en aminokislinski ostanek) se odraža na spremembi valovne dolžine emitirane svetlobe, zato različne vrste kresničk, ki imajo majhne variacije v genskem zapisu za ta protein, oddajajo svetlobo različnih valovnih dolžin (552-582nm, med rumeno in zeleno barvo).

== Viri ==

Worthington Biochemical Corporation - http://www.worthington-biochem.com/lu/default.html

Wapedia - http://wapedia.mobi/sl/Bioluminiscenca

Britannica - http://www.britannica.com/EBchecked/topic/350599/luciferase

Bioluminescence - Chemical Principles and Methods, Osamu Shimomura, World scientific Publishing - 2006

[[Category:LEX]]

Luciferaza

2010-11-05T20:01:25Z

ValterB: /* Luciferaza kresničk */

== Luciferaza ==

Luciferaza[http://en.wikipedia.org/wiki/File:Firefly_Luciferase_Crystal_Structure.rsh.png] je ime za vrsto proteinskih encimov, ki sodelujejo pri reakcijah bioluminiscence v različnih živih bitjih. Običajno se to ime uporablja za encim v kresničkah (''Photinus Pyralis''). Ime je izpeljanka iz latinskih besed lucem ferre, kar pomeni prinašalec luči.

Različne variacije tega encima se nahajajo v družini kresničk in hroščev, v gobah (kot je ''Omphalotus Olearius''[http://en.wikipedia.org/wiki/Omphalotus_olearius]) in veliko morskih organizmih (meduze, sipe, globokomorske ribe, mikroorganizmi). Razvili so se neodvisno in se razlikujejo po sestavi, substratih in valovni dolžini emitirane svetlobe. Prvo reakcijo luciferin-luciferaza je izvedel francoz Horace Raphael Dubois leta 1885. Iz svetilnih organov hroščev iz roda ''Pyrophorus'' je posebej ekstrahiral encim v hladnem, in substrat v vročem, kjer je encim uničen. Ta dva ekstrakta je nato zmešal, in opazoval svetlobo, ki se je pri tem sproščala. Encim in pripadajoč substrat je tudi poimenoval.
Gen za ta encim so že vgradili v genski zapis raznih višjih organizmov, kot sta miš, zajec in krompir.
Encim se da pripeti na določene molekule (na primer protitelesa), in z emitirano svetlobo spremljati njihov položaj.
Uporablja se tudi za merjenje koncentracije ATP v vzorcih, na podoben način pa se uporablja tudi za detekcijo sledov krvi v forenziki.

== Luciferaza kresničk ==

Pri reakciji bioluminiscence kot kofaktorja nastopata [[ATP]] in magnezijev ion, substrat pa je [[luciferin]]. Optimalna temperatura za delovanje encima iz kresničk je 23-25°C, optimalen pH 7.8, molska masa encima pa med 60000 in 62000 Daltoni. Pri bakterijah molska masa znaša okoli 80000 Daltonov, optimalen pH pa je večinoma pod 7.
Encim v prisotnosti ATP in magnezijevega iona katalizira oksidacijo luciferina do hidroperoksidnega intermediata, ki nato ciklizira in pri dekarboksilaciji odda svetlobo. Reakcija ima zelo dober izkoristek, saj je skoraj vsa energija sproščena v obliki svetlobe.
Že zelo majhna sprememba proteinskega encima luciferaze (en aminokislinski ostanek) se odraža na spremembi valovne dolžine emitirane svetlobe, zato različne vrste kresničk, ki imajo majhne variacije v genskem zapisu za ta protein, oddajajo svetlobo različnih valovnih dolžin (552-582nm, med rumeno in zeleno barvo)

== Viri ==

Worthington Biochemical Corporation - http://www.worthington-biochem.com/lu/default.html

Wapedia - http://wapedia.mobi/sl/Bioluminiscenca

Britannica - http://www.britannica.com/EBchecked/topic/350599/luciferase

Bioluminescence - Chemical Principles and Methods, Osamu Shimomura, World scientific Publishing - 2006

[[Category:LEX]]

Luciferaza

2010-11-05T20:00:23Z

ValterB: /* Luciferaza */

== Luciferaza ==

Luciferaza[http://en.wikipedia.org/wiki/File:Firefly_Luciferase_Crystal_Structure.rsh.png] je ime za vrsto proteinskih encimov, ki sodelujejo pri reakcijah bioluminiscence v različnih živih bitjih. Običajno se to ime uporablja za encim v kresničkah (''Photinus Pyralis''). Ime je izpeljanka iz latinskih besed lucem ferre, kar pomeni prinašalec luči.

Različne variacije tega encima se nahajajo v družini kresničk in hroščev, v gobah (kot je ''Omphalotus Olearius''[http://en.wikipedia.org/wiki/Omphalotus_olearius]) in veliko morskih organizmih (meduze, sipe, globokomorske ribe, mikroorganizmi). Razvili so se neodvisno in se razlikujejo po sestavi, substratih in valovni dolžini emitirane svetlobe. Prvo reakcijo luciferin-luciferaza je izvedel francoz Horace Raphael Dubois leta 1885. Iz svetilnih organov hroščev iz roda ''Pyrophorus'' je posebej ekstrahiral encim v hladnem, in substrat v vročem, kjer je encim uničen. Ta dva ekstrakta je nato zmešal, in opazoval svetlobo, ki se je pri tem sproščala. Encim in pripadajoč substrat je tudi poimenoval.
Gen za ta encim so že vgradili v genski zapis raznih višjih organizmov, kot sta miš, zajec in krompir.
Encim se da pripeti na določene molekule (na primer protitelesa), in z emitirano svetlobo spremljati njihov položaj.
Uporablja se tudi za merjenje koncentracije ATP v vzorcih, na podoben način pa se uporablja tudi za detekcijo sledov krvi v forenziki.

== Luciferaza kresničk ==

Pri reakciji bioluminiscence kot kofaktorja nastopata [[ATP]] in magnezijev ion, substrat pa je [[luciferin]]. Optimalna temperatura za delovanje encima iz kresničk je 23-25°C, optimalen pH 7.8, molska masa encima pa med 60000 in 62000 Daltoni. Pri bakterijah molska masa znaša okoli 80000 Daltonov, optimalen pH pa je večinoma pod 7.
Encim v prisotnosti ATP in magnezijevega iona katalizira oksidacijo luciferina do hidroperoksidnega intermediata, ki nato ciklizira in pri dekarboksilaciji odda svetlobo. Reakcija ima zelo dober izkoristek, saj je skoraj vsa energija sproščena v obliki svetlobe.
Že zelo majhna sprememba proteinskega encima luciferaze (en aminokislinski ostanek) se odraža na spremembi valovne dolžine emitirane svetlobe, zato različne vrste kresničk, ki imajo majhne variacije v genskem zapisu za ta protein, oddajajo svetlobo različnih valovnih dolžin (552-582nm, med rumeno in zeleno barvo)

== Viri ==

Worthington Biochemical Corporation - http://www.worthington-biochem.com/lu/default.html

Wapedia - http://wapedia.mobi/sl/Bioluminiscenca

Britannica - http://www.britannica.com/EBchecked/topic/350599/luciferase

Bioluminescence - Chemical Principles and Methods, Osamu Shimomura, World scientific Publishing - 2006

[[Category:LEX]]

Luciferaza

2010-11-05T19:56:09Z

ValterB: New page: == Luciferaza == Luciferaza[http://en.wikipedia.org/wiki/File:Firefly_Luciferase_Crystal_Structure.rsh.png] je ime za vrsto proteinskih encimov, ki sodelujejo pri reakcijah bioluminiscen...

== Luciferaza ==

Luciferaza[http://en.wikipedia.org/wiki/File:Firefly_Luciferase_Crystal_Structure.rsh.png] je ime za vrsto proteinskih encimov, ki sodelujejo pri reakcijah bioluminiscence v različnih živih bitjih. Običajno se to ime uporablja za encim v kresničkah (''Photinus Pyralis''). Ime je izpeljanka iz latinskih besed lucem ferre, kar pomeni prinašalec luči.

Različne variacije tega encima se nahajajo v družini kresničk in hroščev, v gobah (Omphalotus olearius) in veliko morskih organizmih (meduze, sipe, globokomorske ribe, mikroorganizmi). Razvili so se neodvisno in se razlikujejo po sestavi, substratih in valovni dolžini emitirane svetlobe. Prvo reakcijo luciferin-luciferaza je izvedel francoz Horace Raphael Dubois leta 1885. Iz svetilnih organov hroščev iz roda ''Pyrophorus'' je posebej ekstrahiral encim v hladnem, in substrat v vročem, kjer je encim uničen. Ta dva ekstrakta je nato zmešal, in opazoval svetlobo, ki se je pri tem sproščala. Encim in pripadajoč substrat je tudi poimenoval.
Gen za ta encim so že vgradili v genski zapis raznih višjih organizmov, kot sta miš, zajec in krompir.
Encim se da pripeti na določene molekule (na primer protitelesa), in z emitirano svetlobo spremljati njihov položaj.
Uporablja se tudi za merjenje koncentracije ATP v vzorcih, na podoben način pa se uporablja tudi za detekcijo sledov krvi v forenziki.

== Luciferaza kresničk ==

Pri reakciji bioluminiscence kot kofaktorja nastopata [[ATP]] in magnezijev ion, substrat pa je [[luciferin]]. Optimalna temperatura za delovanje encima iz kresničk je 23-25°C, optimalen pH 7.8, molska masa encima pa med 60000 in 62000 Daltoni. Pri bakterijah molska masa znaša okoli 80000 Daltonov, optimalen pH pa je večinoma pod 7.
Encim v prisotnosti ATP in magnezijevega iona katalizira oksidacijo luciferina do hidroperoksidnega intermediata, ki nato ciklizira in pri dekarboksilaciji odda svetlobo. Reakcija ima zelo dober izkoristek, saj je skoraj vsa energija sproščena v obliki svetlobe.
Že zelo majhna sprememba proteinskega encima luciferaze (en aminokislinski ostanek) se odraža na spremembi valovne dolžine emitirane svetlobe, zato različne vrste kresničk, ki imajo majhne variacije v genskem zapisu za ta protein, oddajajo svetlobo različnih valovnih dolžin (552-582nm, med rumeno in zeleno barvo)

== Viri ==

Worthington Biochemical Corporation - http://www.worthington-biochem.com/lu/default.html

Wapedia - http://wapedia.mobi/sl/Bioluminiscenca

Britannica - http://www.britannica.com/EBchecked/topic/350599/luciferase

Bioluminescence - Chemical Principles and Methods, Osamu Shimomura, World scientific Publishing - 2006

[[Category:LEX]]