Wiki FKKT - User contributions [en]

2015-bionano-seminar

2015-04-04T12:58:55Z

MitjaCrcek:

= 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||||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||||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||||12.04.||14.04.||Katja Lovrin||Veronika Jarc
|-
| Vida Špindler||Jernej Pušnik||||12.04.||14.04.||Saša Balažic||Iza Ogris
|-
| Jasmina Sedmak||Maxi Sagmeister||||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||||03.05.||05.05.||Vida Špindler||Kim Kos
|-
| Simon Preložnik||Maja Remškar||||03.05.||05.05.||Jasmina Sedmak||Estera Merljak
|-
| Kaja Javoršek||Tina Gregorič||||03.05.||05.05.||Sanja Popović||Jernej Pušnik
|-
| Damir Hamulić||Anita Kustec||||10.05.||12.05.||Blaž Komar||Maxi Sagmeister
|-
| Janja Fortin||Tina Snoj||||10.05.||12.05.||Blaž Perič||Benjamin Bajželj
|-
| Rajko Vnuk||Mojca Banič||||10.05.||12.05.||Simon Preložnik||Alja Zottel
|-
| Rok Grm||Ajda Rojc||||17.05.||19.05.||Kaja Javoršek||Katarina Uršič
|-
| Kristina Gavranić||Barbara Žužek||||17.05.||19.05.||Damir Hamulić||Maja Remškar
|-
| Urška Mohorič||Griša Prinčič||||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||||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
|-
| Aneja Tuljak||Tjaša Goričan||||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.

==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.

Bioremediacija s pesticidi okužene vode z uporabo encima, ki razgrajuje organofosfate in je vezan na netkan poliestrski tekstil

2015-03-29T18:59:35Z

MitjaCrcek:

'''Uvod'''

Organofosfati ali fosfatni estri so organske molekule in mnoge izmed njih so ključnega pomena za naše življenje (DNA, RNA, kofaktorji) [1]. V biotehnologiji, zdravstvu in agrikulturi se izraz organofosfati nanaša na skupino insekticidov, ki delujejo na encim acetil-holinesterazo. Omenjeni encim ima ključno vlogo pri prenosu signalov v živčnem sistemu insektov, ljudi in mnogih drugih živali. Organofosfatni pesticidi, kot sta na primer paration in metil paration (MP), ireverzibilno inaktivirajo acetil-holinesterazo in so zato učinkoviti pesticidi [1]. V primerjavi z organokloridi se v naravi hitreje razgradijo in so zato manj nevarni za človeka, vseeno pa lahko povzročijo hujše bolezni. Zato se je pojavila potreba po razvoju bioremediacijskega sistema za razgradnjo organofosfatov v okolju, kjer uporabljajo omenjene insekticide.

=='''Ideja in cilji raziskave'''==

Imobilizacija encimov ima mnoge prednosti pred uporabo topnih encimov in ima zato velik interes v industriji. Poleg tega je bioremediacija vse pomembnejša tema okoljevarstvenikov, zato so se v raziskavi osredotočili na imobilizacijo encima '''OpdA''' (Organophosphate degrading enzyme A), imenovanega tudi fosfotriesteraza ali organofosforna hidrolaza (iz bakterije ''Pseudomonas diminuta''). Omenjeni encim ima široko substratno specifičnost; cepi lahko vezi P-O, P-CN in P-F v mnogih organofosfatnih spojinah. Katalizira tudi pretvorbo toksičnega metil parationa (MP) v manj toksičen p-nitrofenol (pNP), ki absorbira, zato je primeren za uporabo v raziskavah [2,3]. V raziskavi so preverili, kako vezava encima na poliestrska vlakna vpliva na njegove kinetične lastnosti, stabilnost in ponovno uporabnost.

=='''Potek raziskave in rezultati'''==

Imobilizacijski proces je vseboval tri stopnje. V prvi fazi so poliestrska vlakna tretirali z 99% EDA. Ena od aminskih skupin EDA-ja je reagirala s poliestrskim vlaknom, druga aminska skupina pa je ostala prosta. V drugem koraku so uporabili povezovalno molekulo glutaraldehid, ki se je vezal na prosto aminsko skupino EDA-ja. V zadnji stopnji so dodali očiščen encim OpdA, ki se je z N-koncem vezal na glutaraldehid.

Po pripravi imobiliziranega encima so opravili kinetične meritve, pri čemer so opazovali različne parametre. Pričakovano se je povečala Km encima (za substrat MP) in sicer za 2x v primerjavi s topnim encimom. Glede na povprečno povečanje konstante pri imobilizaciji encimov (3-6x) lahko rečemo, da je bila imobilizacija zelo uspešna. Ugotovili so tudi, da lahko imobiliziran encim OpdA v primerjavi s topnim encimom deluje v precej širšem pH območju, saj je topen encim praktično neaktiven že pri pH-jih manjših od 6,5, medtem ko imobiliziran encim še pri pH=5 ohrani polovico svoje aktivnosti. Imobiliziran encim je prav tako termostabilnejši in tudi po večurni inkubaciji pri 55 °C ohrani praktično celotno aktivnost. Testirali so tudi različne pogoje shranjevanja imobiliziranega encima in ugotovili, da je najustreznejše shranjevanje pri 4 °C v suhi obliki ali v puferski raztopini.

Sledilo je testiranje učinkovitosti pripravljenega imobiliziranega encima pri razgradnji metil parationa. Dokazali so, da ob ustrezni količini poliestrskih vlaken z encimom razpade praktično ves MP že pri 30-60 minutah. Ugotovili so, da encim po večkratni uporabi delno izgubi svojo aktivnost in potrebuje več časa, da razgradi enako količino MP-ja. To pripisujejo encimski nestabilnosti in njegovi proteolizi. Pripravili so tudi kolone z enakim poliestrskim nosilcem, na katerega so vezali OpdA in ugotovili, da se pri nižjih pretokih razgradi praktično ves metil paration. V primeru velikega pretoka in nanosa večje količine raztopine MP-ja na kolono, pa ne pride do popolne razgradnje MP-ja. Encim v kolonah je precej obstojen približno 2 meseca, nato pa naj bi izgubljal zmožnost degradacije organofosfatov.

Preverili so tudi razgradnjo kumafosa, ki se prav tako uvršča med organofosfatne insekticide. Ugotovili so, da lahko pripravljen sistem uporabijo tudi za razgrajevanje omenjenega organofosfata, precej verjetno pa tudi za ostale organofosfatne insekticide.

=='''Zaključek'''==

V okviru raziskave so znanstveniki pokazali, da je netkan poliestrski tekstil (oz. poliestrska vlakna) ustrezen nosilec za bioremediacijske encime, ki razgrajujejo organofosfate. Imobiliziran encim kaže povišano stabilnost in nizko izgubo aktivnosti v primerjavi s topnim encimom. Prav tako izkazuje sposobnost razgradnje organofosfatnih pesticidov že pri nizkih koncentracijah organofosfatov, uporablja pa se lahko v kontinuirnih ali ne-kontinuirnih procesih.

=='''Viri in literatura'''==

Izhodiščni članek: Y. Gao, Y. B. Truong, P. Cacioli, P. Butler, in I. L. Kyratzis, „Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles“, Enzyme Microb. Technol., let. 54, str. 38–44, jan. 2014.

[1] „Organophosphate“, Wikipedia, the free encyclopedia. 25-mar-2015.

[2] F. M. Raushel, „Bacterial detoxification of organophosphate nerve agents“, Curr. Opin. Microbiol., let. 5, št. 3, str. 288–295, jun. 2002.

[3] K. Lai, N. J. Stolowich, in J. R. Wild, „Characterization of P-S Bond Hydrolysis in Organophosphorothioate Pesticides by Organophosphorus Hydrolase“, Arch. Biochem. Biophys., let. 318, št. 1, str. 59–64, maj 1995.

Bioremediacija s pesticidi okužene vode z uporabo encima, ki razgrajuje organofosfate in je vezan na netkan poliestrski tekstil

2015-03-29T18:58:20Z

MitjaCrcek:

'''Uvod'''

Organofosfati ali fosfatni estri so organske molekule in mnoge izmed njih so ključnega pomena za naše življenje (DNA, RNA, kofaktorji) [1]. V biotehnologiji, zdravstvu in agrikulturi se izraz organofosfati nanaša na skupino insekticidov, ki delujejo na encim acetil-holinesterazo. Omenjeni encim ima ključno vlogo pri prenosu signalov v živčnem sistemu insektov, ljudi in mnogih drugih živali. Organofosfatni pesticidi, kot sta na primer paration in metil paration (MP), ireverzibilno inaktivirajo acetil-holinesterazo in so zato učinkoviti pesticidi [1]. V primerjavi z organokloridi se v naravi hitreje razgradijo in so zato manj nevarni za človeka, vseeno pa lahko povzročijo hujše bolezni. Zato se je pojavila potreba po razvoju bioremediacijskega sistema za razgradnjo organofosfatov v okolju, kjer uporabljajo omenjene insekticide.

=='''Ideja in cilji raziskave'''==

Imobilizacija encimov ima mnoge prednosti pred uporabo topnih encimov in ima zato velik interes v industriji. Poleg tega je bioremediacija vse pomembnejša tema okoljevarstvenikov, zato so se v raziskavi osredotočili na imobilizacijo encima '''OpdA''' (Organophosphate degrading enzyme A), imenovanega tudi fosfotriesteraza ali organofosforna hidrolaza (iz bakterije ''Pseudomonas diminuta''). Omenjeni encim ima široko substratno specifičnost; cepi lahko vezi P-O, P-CN in P-F v mnogih organofosfatnih spojinah. Katalizira tudi pretvorbo toksičnega metil parationa (MP) v manj toksičen p-nitrofenol (pNP), ki absorbira, zato je primeren za uporabo v raziskavah [2,3]. V raziskavi so preverili, kako vezava encima na poliestrska vlakna vpliva na njegove kinetične lastnosti, stabilnost in ponovno uporabnost.

=='''Potek raziskave in rezultati'''==

Imobilizacijski proces je vseboval tri stopnje. V prvi fazi so poliestrska vlakna tretirali z 99% EDA. Ena od aminskih skupin EDA-ja je reagirala s poliestrskim vlaknom, druga aminska skupina pa je ostala prosta. V drugem koraku so uporabili povezovalno molekulo glutaraldehid, ki se je vezal na prosto aminsko skupino EDA-ja. V zadnji stopnji so dodali očiščen encim OpdA, ki se je z N-koncem vezal na glutaraldehid.

Po pripravi imobiliziranega encima so opravili kinetične meritve, pri čemer so opazovali različne parametre. Pričakovano se je povečala Km encima (za substrat MP) in sicer za 2x v primerjavi s topnim encimom. Glede na povprečno povečanje konstante pri imobilizaciji encimov (3-6x) lahko rečemo, da je bila imobilizacija zelo uspešna. Ugotovili so tudi, da lahko imobiliziran encim OpdA v primerjavi s topnim encimom deluje v precej širšem pH območju, saj je topen encim praktično neaktiven že pri pH-jih manjših od 6,5, medtem ko imobiliziran encim še pri pH=5 ohrani polovico svoje aktivnosti. Imobiliziran encim je prav tako termostabilnejši in tudi po večurni inkubaciji pri 55 °C ohrani praktično celotno aktivnost. Testirali so tudi različne pogoje shranjevanja imobiliziranega encima in ugotovili, da je najustreznejše shranjevanje pri 4 °C v suhi obliki ali v puferski raztopini. µ

Sledilo je testiranje učinkovitosti pripravljenega imobiliziranega encima pri razgradnji metil parationa. Dokazali so, da ob ustrezni količini poliestrskih vlaken z encimom razpade praktično ves MP že pri 30-60 minutah. Ugotovili so, da encim po večkratni uporabi delno izgubi svojo aktivnost in potrebuje več časa, da razgradi enako količino MP-ja. To pripisujejo encimski nestabilnosti in njegovi proteolizi. Pripravili so tudi kolone z enakim poliestrskim nosilcem, na katerega so vezali OpdA in ugotovili, da se pri nižjih pretokih razgradi praktično ves metil paration. V primeru velikega pretoka in nanosa večje količine raztopine MP-ja na kolono, pa ne pride do popolne razgradnje MP-ja. Encim v kolonah je precej obstojen približno 2 meseca, nato pa naj bi izgubljal zmožnost degradacije organofosfatov.

Preverili so tudi razgradnjo kumafosa, ki se prav tako uvršča med organofosfatne insekticide. Ugotovili so, da lahko pripravljen sistem uporabijo tudi za razgrajevanje omenjenega organofosfata, precej verjetno pa tudi za ostale organofosfatne insekticide.

=='''Zaključek'''==

V okviru raziskave so znanstveniki pokazali, da je netkan poliestrski tekstil (oz. poliestrska vlakna) ustrezen nosilec za bioremediacijske encime, ki razgrajujejo organofosfate. Imobiliziran encim kaže povišano stabilnost in nizko izgubo aktivnosti v primerjavi s topnim encimom. Prav tako izkazuje sposobnost razgradnje organofosfatnih pesticidov že pri nizkih koncentracijah organofosfatov, uporablja pa se lahko v kontinuirnih ali ne-kontinuirnih procesih.

=='''Viri in literatura'''==

Izhodiščni članek: Y. Gao, Y. B. Truong, P. Cacioli, P. Butler, in I. L. Kyratzis, „Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles“, Enzyme Microb. Technol., let. 54, str. 38–44, jan. 2014.

[1] „Organophosphate“, Wikipedia, the free encyclopedia. 25-mar-2015.

[2] F. M. Raushel, „Bacterial detoxification of organophosphate nerve agents“, Curr. Opin. Microbiol., let. 5, št. 3, str. 288–295, jun. 2002.

[3] K. Lai, N. J. Stolowich, in J. R. Wild, „Characterization of P-S Bond Hydrolysis in Organophosphorothioate Pesticides by Organophosphorus Hydrolase“, Arch. Biochem. Biophys., let. 318, št. 1, str. 59–64, maj 1995.

Bioremediacija s pesticidi okužene vode z uporabo encima, ki razgrajuje organofosfate in je vezan na netkan poliestrski tekstil

2015-03-29T18:57:48Z

MitjaCrcek:

'''Uvod'''

Organofosfati ali fosfatni estri so organske molekule in mnoge izmed njih so ključnega pomena za naše življenje (DNA, RNA, kofaktorji) [1]. V biotehnologiji, zdravstvu in agrikulturi se izraz organofosfati nanaša na skupino insekticidov, ki delujejo na encim acetil-holinesterazo. Omenjeni encim ima ključno vlogo pri prenosu signalov v živčnem sistemu insektov, ljudi in mnogih drugih živali. Organofosfatni pesticidi, kot sta na primer paration in metil paration (MP), ireverzibilno inaktivirajo acetil-holinesterazo in so zato učinkoviti pesticidi [1]. V primerjavi z organokloridi se v naravi hitreje razgradijo in so zato manj nevarni za človeka, vseeno pa lahko povzročijo hujše bolezni. Zato se je pojavila potreba po razvoju bioremediacijskega sistema za razgradnjo organofosfatov v okolju, kjer uporabljajo omenjene insekticide.

=='''Ideja in cilji raziskave'''==

Imobilizacija encimov ima mnoge prednosti pred uporabo topnih encimov in ima zato velik interes v industriji. Poleg tega je bioremediacija vse pomembnejša tema okoljevarstvenikov, zato so se v raziskavi osredotočili na imobilizacijo encima '''OpdA''' (Organophosphate degrading enzyme A), imenovanega tudi fosfotriesteraza ali organofosforna hidrolaza (iz bakterije ''Pseudomonas diminuta''). Omenjeni encim ima široko substratno specifičnost; cepi lahko vezi P-O, P-CN in P-F v mnogih organofosfatnih spojinah. Katalizira tudi pretvorbo toksičnega metil parationa (MP) v manj toksičen p-nitrofenol (pNP), ki absorbira, zato je primeren za uporabo v raziskavah [2][3]. V raziskavi so preverili, kako vezava encima na poliestrska vlakna vpliva na njegove kinetične lastnosti, stabilnost in ponovno uporabnost.

=='''Potek raziskave in rezultati'''==

Imobilizacijski proces je vseboval tri stopnje. V prvi fazi so poliestrska vlakna tretirali z 99% EDA. Ena od aminskih skupin EDA-ja je reagirala s poliestrskim vlaknom, druga aminska skupina pa je ostala prosta. V drugem koraku so uporabili povezovalno molekulo glutaraldehid, ki se je vezal na prosto aminsko skupino EDA-ja. V zadnji stopnji so dodali očiščen encim OpdA, ki se je z N-koncem vezal na glutaraldehid.

Po pripravi imobiliziranega encima so opravili kinetične meritve, pri čemer so opazovali različne parametre. Pričakovano se je povečala Km encima (za substrat MP) in sicer za 2x v primerjavi s topnim encimom. Glede na povprečno povečanje konstante pri imobilizaciji encimov (3-6x) lahko rečemo, da je bila imobilizacija zelo uspešna. Ugotovili so tudi, da lahko imobiliziran encim OpdA v primerjavi s topnim encimom deluje v precej širšem pH območju, saj je topen encim praktično neaktiven že pri pH-jih manjših od 6,5, medtem ko imobiliziran encim še pri pH=5 ohrani polovico svoje aktivnosti. Imobiliziran encim je prav tako termostabilnejši in tudi po večurni inkubaciji pri 55 °C ohrani praktično celotno aktivnost. Testirali so tudi različne pogoje shranjevanja imobiliziranega encima in ugotovili, da je najustreznejše shranjevanje pri 4 °C v suhi obliki ali v puferski raztopini. µ

Sledilo je testiranje učinkovitosti pripravljenega imobiliziranega encima pri razgradnji metil parationa. Dokazali so, da ob ustrezni količini poliestrskih vlaken z encimom razpade praktično ves MP že pri 30-60 minutah. Ugotovili so, da encim po večkratni uporabi delno izgubi svojo aktivnost in potrebuje več časa, da razgradi enako količino MP-ja. To pripisujejo encimski nestabilnosti in njegovi proteolizi. Pripravili so tudi kolone z enakim poliestrskim nosilcem, na katerega so vezali OpdA in ugotovili, da se pri nižjih pretokih razgradi praktično ves metil paration. V primeru velikega pretoka in nanosa večje količine raztopine MP-ja na kolono, pa ne pride do popolne razgradnje MP-ja. Encim v kolonah je precej obstojen približno 2 meseca, nato pa naj bi izgubljal zmožnost degradacije organofosfatov.

Preverili so tudi razgradnjo kumafosa, ki se prav tako uvršča med organofosfatne insekticide. Ugotovili so, da lahko pripravljen sistem uporabijo tudi za razgrajevanje omenjenega organofosfata, precej verjetno pa tudi za ostale organofosfatne insekticide.

=='''Zaključek'''==

V okviru raziskave so znanstveniki pokazali, da je netkan poliestrski tekstil (oz. poliestrska vlakna) ustrezen nosilec za bioremediacijske encime, ki razgrajujejo organofosfate. Imobiliziran encim kaže povišano stabilnost in nizko izgubo aktivnosti v primerjavi s topnim encimom. Prav tako izkazuje sposobnost razgradnje organofosfatnih pesticidov že pri nizkih koncentracijah organofosfatov, uporablja pa se lahko v kontinuirnih ali ne-kontinuirnih procesih.

=='''Viri in literatura'''==

Izhodiščni članek: Y. Gao, Y. B. Truong, P. Cacioli, P. Butler, in I. L. Kyratzis, „Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles“, Enzyme Microb. Technol., let. 54, str. 38–44, jan. 2014.

[1] „Organophosphate“, Wikipedia, the free encyclopedia. 25-mar-2015.

[2] F. M. Raushel, „Bacterial detoxification of organophosphate nerve agents“, Curr. Opin. Microbiol., let. 5, št. 3, str. 288–295, jun. 2002.

[3] K. Lai, N. J. Stolowich, in J. R. Wild, „Characterization of P-S Bond Hydrolysis in Organophosphorothioate Pesticides by Organophosphorus Hydrolase“, Arch. Biochem. Biophys., let. 318, št. 1, str. 59–64, maj 1995.

Bioremediacija s pesticidi okužene vode z uporabo encima, ki razgrajuje organofosfate in je vezan na netkan poliestrski tekstil

2015-03-29T18:56:18Z

MitjaCrcek:

'''Uvod'''

Organofosfati ali fosfatni estri so organske molekule in mnoge izmed njih so ključnega pomena za naše življenje (DNA, RNA, kofaktorji) [1]. V biotehnologiji, zdravstvu in agrikulturi se izraz organofosfati nanaša na skupino insekticidov, ki delujejo na encim acetil-holinesterazo. Omenjeni encim ima ključno vlogo pri prenosu signalov v živčnem sistemu insektov, ljudi in mnogih drugih živali. Organofosfatni pesticidi, kot sta na primer paration in metil paration (MP), ireverzibilno inaktivirajo acetil-holinesterazo in so zato učinkoviti pesticidi [1]. V primerjavi z organokloridi se v naravi hitreje razgradijo in so zato manj nevarni za človeka, vseeno pa lahko povzročijo hujše bolezni. Zato se je pojavila potreba po razvoju bioremediacijskega sistema za razgradnjo organofosfatov v okolju, kjer uporabljajo omenjene insekticide.

=='''Ideja in cilji raziskave'''==

Imobilizacija encimov ima mnoge prednosti pred uporabo topnih encimov in ima zato velik interes v industriji. Poleg tega je bioremediacija vse pomembnejša tema okoljevarstvenikov, zato so se v raziskavi osredotočili na imobilizacijo encima OpdA (Organophosphate degrading enzyme A), imenovanega tudi fosfotriesteraza ali organofosforna hidrolaza (iz bakterije ''Pseudomonas diminuta''). Omenjeni encim ima široko substratno specifičnost; cepi lahko vezi P-O, P-CN in P-F v mnogih organofosfatnih spojinah. Katalizira tudi pretvorbo toksičnega metil parationa (MP) v manj toksičen p-nitrofenol (pNP), ki absorbira, zato je primeren za uporabo v raziskavah [2][3]. V raziskavi so preverili, kako vezava encima na poliestrska vlakna vpliva na njegove kinetične lastnosti, stabilnost in ponovno uporabnost.

=='''Potek raziskave in rezultati'''==

Imobilizacijski proces je vseboval tri stopnje. V prvi fazi so poliestrska vlakna tretirali z 99% EDA. Ena od aminskih skupin EDA-ja je reagirala s poliestrskim vlaknom, druga aminska skupina pa je ostala prosta. V drugem koraku so uporabili povezovalno molekulo glutaraldehid, ki se je vezal na prosto aminsko skupino EDA-ja. V zadnji stopnji so dodali očiščen encim OpdA, ki se je z N-koncem vezal na glutaraldehid.

Po pripravi imobiliziranega encima so opravili kinetične meritve, pri čemer so opazovali različne parametre. Pričakovano se je povečala Km encima (za substrat MP) in sicer za 2x v primerjavi s topnim encimom. Glede na povprečno povečanje konstante pri imobilizaciji encimov (3-6x) lahko rečemo, da je bila imobilizacija zelo uspešna. Ugotovili so tudi, da lahko imobiliziran encim OpdA v primerjavi s topnim encimom deluje v precej širšem pH območju, saj je topen encim praktično neaktiven že pri pH-jih manjših od 6,5, medtem ko imobiliziran encim še pri pH=5 ohrani polovico svoje aktivnosti. Imobiliziran encim je prav tako termostabilnejši in tudi po večurni inkubaciji pri 55 °C ohrani praktično celotno aktivnost. Testirali so tudi različne pogoje shranjevanja imobiliziranega encima in ugotovili, da je najustreznejše shranjevanje pri 4 °C v suhi obliki ali v puferski raztopini. µ

Sledilo je testiranje učinkovitosti pripravljenega imobiliziranega encima pri razgradnji metil parationa. Dokazali so, da ob ustrezni količini poliestrskih vlaken z encimom razpade praktično ves MP že pri 30-60 minutah. Ugotovili so, da encim po večkratni uporabi delno izgubi svojo aktivnost in potrebuje več časa, da razgradi enako količino MP-ja. To pripisujejo encimski nestabilnosti in njegovi proteolizi. Pripravili so tudi kolone z enakim poliestrskim nosilcem, na katerega so vezali OpdA in ugotovili, da se pri nižjih pretokih razgradi praktično ves metil paration. V primeru velikega pretoka in nanosa večje količine raztopine MP-ja na kolono, pa ne pride do popolne razgradnje MP-ja. Encim v kolonah je precej obstojen približno 2 meseca, nato pa naj bi izgubljal zmožnost degradacije organofosfatov.

Preverili so tudi razgradnjo kumafosa, ki se prav tako uvršča med organofosfatne insekticide. Ugotovili so, da lahko pripravljen sistem uporabijo tudi za razgrajevanje omenjenega organofosfata, precej verjetno pa tudi za ostale organofosfatne insekticide.

=='''Zaključek'''==

V okviru raziskave so znanstveniki pokazali, da je netkan poliestrski tekstil (oz. poliestrska vlakna) ustrezen nosilec za bioremediacijske encime, ki razgrajujejo organofosfate. Imobiliziran encim kaže povišano stabilnost in nizko izgubo aktivnosti v primerjavi s topnim encimom. Prav tako izkazuje sposobnost razgradnje organofosfatnih pesticidov že pri nizkih koncentracijah organofosfatov, uporablja pa se lahko v kontinuirnih ali ne-kontinuirnih procesih.

=='''Viri in literatura'''==

Izhodiščni članek: Y. Gao, Y. B. Truong, P. Cacioli, P. Butler, in I. L. Kyratzis, „Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles“, Enzyme Microb. Technol., let. 54, str. 38–44, jan. 2014.

[1] „Organophosphate“, Wikipedia, the free encyclopedia. 25-mar-2015.

[2] F. M. Raushel, „Bacterial detoxification of organophosphate nerve agents“, Curr. Opin. Microbiol., let. 5, št. 3, str. 288–295, jun. 2002.

[3] K. Lai, N. J. Stolowich, in J. R. Wild, „Characterization of P-S Bond Hydrolysis in Organophosphorothioate Pesticides by Organophosphorus Hydrolase“, Arch. Biochem. Biophys., let. 318, št. 1, str. 59–64, maj 1995.

Bioremediacija s pesticidi okužene vode z uporabo encima, ki razgrajuje organofosfate in je vezan na netkan poliestrski tekstil

2015-03-29T18:53:00Z

MitjaCrcek: New page: '''Uvod''' Organofosfati ali fosfatni estri so organske molekule in mnoge izmed njih so ključnega pomena za naše življenje (DNA, RNA, kofaktorji) [1]. V biotehnologiji, zdravstvu in ag...

'''Uvod'''

Organofosfati ali fosfatni estri so organske molekule in mnoge izmed njih so ključnega pomena za naše življenje (DNA, RNA, kofaktorji) [1]. V biotehnologiji, zdravstvu in agrikulturi se izraz organofosfati nanaša na skupino insekticidov, ki delujejo na encim acetil-holinesterazo. Omenjeni encim ima ključno vlogo pri prenosu signalov v živčnem sistemu insektov, ljudi in mnogih drugih živali. Organofosfatni pesticidi, kot sta na primer paration in metil paration (MP), ireverzibilno inaktivirajo acetil-holinesterazo in so zato učinkoviti pesticidi [1]. V primerjavi z organokloridi se v naravi hitreje razgradijo in so zato manj nevarni za človeka, vseeno pa lahko povzročijo hujše bolezni. Zato se je pojavila potreba po razvoju bioremediacijskega sistema za razgradnjo organofosfatov v okolju, kjer uporabljajo omenjene insekticide.

=='''Ideja in cilji raziskave'''==

Imobilizacija encimov ima mnoge prednosti pred uporabo topnih encimov in ima zato velik interes v industriji. Poleg tega je bioremediacija vse pomembnejša tema okoljevarstvenikov, zato so se v raziskavi osredotočili na imobilizacijo encima OpdA (Organophosphate degrading enzyme A), imenovanega tudi fosfotriesteraza ali organofosforna hidrolaza (iz bakterije ''Pseudomonas diminuta''). Omenjeni encim ima široko substratno specifičnost; cepi lahko vezi P-O, P-CN in P-F v mnogih organofosfatnih spojinah. Katalizira tudi pretvorbo toksičnega metil parationa (MP) v manj toksičen p-nitrofenol (pNP), ki absorbira, zato je primeren za uporabo v raziskavah [2][3]. V raziskavi so preverili, kako vezava encima na poliestrska vlakna vpliva na njegove kinetične lastnosti, stabilnost in ponovno uporabnost.

=='''Potek raziskave in rezultati'''==

Imobilizacijski proces je vseboval tri stopnje. V prvi fazi so poliestrska vlakna tretirali z 99% EDA. Ena od aminskih skupin EDA-ja je reagirala s poliestrskim vlaknom, druga aminska skupina pa je ostala prosta. V drugem koraku so uporabili povezovalno molekulo glutaraldehid, ki se je vezal na prosto aminsko skupino EDA-ja. V zadnji stopnji so dodali očiščen encim OpdA, ki se je z N-koncem vezal na glutaraldehid.

Po pripravi imobiliziranega encima so opravili kinetične meritve, pri čemer so opazovali različne parametre. Pričakovano se je povečala Km encima (za substrat MP) in sicer za 2x v primerjavi s topnim encimom. Glede na povprečno povečanje konstante pri imobilizaciji encimov (3-6x) lahko rečemo, da je bila imobilizacija zelo uspešna. Ugotovili so tudi, da lahko imobiliziran encim OpdA v primerjavi s topnim encimom deluje v precej širšem pH območju, saj je topen encim praktično neaktiven že pri pH-jih manjših od 6,5, medtem ko imobiliziran encim še pri pH=5 ohrani polovico svoje aktivnosti. Imobiliziran encim je prav tako termostabilnejši in tudi po večurni inkubaciji pri 55 °C ohrani praktično celotno aktivnost. Testirali so tudi različne pogoje shranjevanja imobiliziranega encima in ugotovili, da je najustreznejše shranjevanje pri 4 °C v suhi obliki ali v puferski raztopini. µ

Sledilo je testiranje učinkovitosti pripravljenega imobiliziranega encima pri razgradnji metil parationa. Dokazali so, da ob ustrezni količini poliestrskih vlaken z encimom razpade praktično ves MP že pri 30-60 minutah. Ugotovili so, da encim po večkratni uporabi delno izgubi svojo aktivnost in potrebuje več časa, da razgradi enako količino MP-ja. To pripisujejo encimski nestabilnosti in njegovi proteolizi. Pripravili so tudi kolone z enakim poliestrskim nosilcem, na katerega so vezali OpdA in ugotovili, da se pri nižjih pretokih razgradi praktično ves metil paration. V primeru velikega pretoka in nanosa večje količine raztopine MP-ja na kolono, pa ne pride do popolne razgradnje MP-ja. Encim v kolonah je precej obstojen približno 2 meseca, nato pa naj bi izgubljal zmožnost degradacije organofosfatov.

Preverili so tudi razgradnjo kumafosa, ki se prav tako uvršča med organofosfatne insekticide. Ugotovili so, da lahko pripravljen sistem uporabijo tudi za razgrajevanje omenjenega organofosfata, precej verjetno pa tudi za ostale organofosfatne insekticide.

=='''Zaključek'''==

V okviru raziskave so znanstveniki pokazali, da je netkan poliestrski tekstil (oz. poliestrska vlakna) ustrezen nosilec za bioremediacijske encime, ki razgrajujejo organofosfate. Imobiliziran encim kaže povišano stabilnost in nizko izgubo aktivnosti v primerjavi s topnim encimom. Prav tako izkazuje sposobnost razgradnje organofosfatnih pesticidov že pri nizkih koncentracijah organofosfatov, uporablja pa se lahko v kontinuirnih ali ne-kontinuirnih procesih.

=='''Literatura'''==

Izhodiščni članek: Y. Gao, Y. B. Truong, P. Cacioli, P. Butler, in I. L. Kyratzis, „Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles“, Enzyme Microb. Technol., let. 54, str. 38–44, jan. 2014.

[1] „Organophosphate“, Wikipedia, the free encyclopedia. 25-mar-2015.

[2] F. M. Raushel, „Bacterial detoxification of organophosphate nerve agents“, Curr. Opin. Microbiol., let. 5, št. 3, str. 288–295, jun. 2002.

[3] K. Lai, N. J. Stolowich, in J. R. Wild, „Characterization of P-S Bond Hydrolysis in Organophosphorothioate Pesticides by Organophosphorus Hydrolase“, Arch. Biochem. Biophys., let. 318, št. 1, str. 59–64, maj 1995.

MBT seminarji 2015

2015-03-29T07:36:58Z

MitjaCrcek:

'''Seznam seminarjev iz Molekularne biotehnologije v študijskem letu 2014/15'''

Tabela za razpored po tednih bo objavljena v spletni učilnici, vanjo pa se vpišite tudi za kratke predstavitve novic (3 min, dvakrat v semestru). 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 (ponedeljek oz. torek). Angleški naslov prevedite tudi v slovenščino - to bo naslov povzetka, ki ga objavite na posebni strani, tako kot so to naredili kolegi pred vami (oz. lani).

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 povežite z novo stranjo, na kateri bo povzetek)

'''Naslovi odobrenih člankov po temah:'''

'''Gensko spremenjene rastline''' 

# Successful high-level accumulation of fish oil omega-3 long-chain polyunsaturated fatty acids in a transgenic oilseed crop (Ruiz-Lopez, N., et al; The plant journal 77, 198-208, 2014; http://www.ncbi.nlm.nih.gov/pubmed/24308505). [[Uspešna priprava gensko spremenjene oljne rastline z visoko vsebnostjo omega-3 polinenasičenih maščobnih kislin.]] Petra Malavašič, 20. marca 2015
#A simpliﬁed and accurate detection of the genetically modiﬁed wheat MON71800 with one calibrator plasmid (Jae Juan, S.,et al; Food Chemistry 176, 1-6, ;http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S03088146140196572015 [[Poenostavljena in točna detekcija gensko spemenjene pšenice MON71800 z enim kalibratorskim plazmidom]] Matej Lesar, 20.marca 2015

'''Gensko spremenjene živali''' 

# [[A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expresion]] (H. Chen; et all.; BMC Biotechnology 2015, 15:4, doi:10.1186/s12896-015-0121-4; http://www.biomedcentral.com/1472-6750/15/4). Edvinas Grauželis, 27. marca 2015 (in English)
# Production of functional active human growth factors in insects used as living biofactories (B. Dudognon, et al; Journal of Biotechnology 184, 229–239, 2014; http://dx.doi.org/10.1016/j.jbiotec.2014.05.030). [[Proizvodnja funkcionalno aktivnih človeških rastnih faktorjev v insektih uporabljenih kot žive biotovarne]] Maxi Sagmeister, 27. marca 2015

'''Okolje''' 
# Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles (Yuan Gao ''et al.'', Enzyme and Microbial Technology, vol. 54, pages 38-44, 10.1.2014, http://www.sciencedirect.com/science/article/pii/S0141022913002044). [[Bioremediacija s pesticidi okužene vode z uporabo encima, ki razgrajuje organofosfate in je vezan na netkan poliestrski tekstil]]. Mitja Crček, 3. aprila 2015
# Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene (A. W. Vail ''et al.''; Transgenic Research, 29. 11. 2014; http://link.springer.com/article/10.1007/s11248-014-9851-7). Biorazgradnja atrazina s tremi transgenskimi travami in alfalfo, ki izražajo gen za modificirano bakterijsko atrazin klorohidrolazo. Mirjam Kmetič, 3. aprila 2015

'''Terapevtiki''' 
# Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant (M. Folcher; Nature Communications 5, 1–11, 2014; http://www.nature.com/ncomms/2014/141111/ncomms6392/full/ncomms6392.html) Z EEG nadzorovano izražanje transgena preko brezžično napajanega optogenetskega celičnega vsadka. Luka Smole, 10. aprila 2015

'''Encimi''' 

# Construction of efficient xylose utilizing ''Pichia pastoris'' for industrial enzyme production (Li ''et al''; Microbial Cell Factories 14:22, 1-10, 2015; http://www.microbialcellfactories.com/content/14/1/22). Priprava ''Pichie pastoris'', ki učinkovito uporablja ksilozo, za industrijsko proizvodnjo encimov. Špela Tomaž, 17. aprila 2015

'''Protitelesa''' 

# Functional mutations in and characterization of VHH against Helicobacter pylori urease (R. Hoseinpoor ''et al''; Applied Biochemistry and Biotechnology 172, 3079-3091, 2014; http://link.springer.com/article/10.1007/s12010-014-0750-4). Funkcionalne mutacije in karakterizacija VHH proti ureazi ''Helicobacter pylori''. Marko Radojković, 24. aprila 2015
#Ethanol precipitation for purification of recombinant antibodies (A. Tscheliessnig ''et al''; Journal of Biotechnology 188, 17-28, 2014; http://www.sciencedirect.com/science/article/pii/S0168165614007810). Čiščenje rekombinantnih protiteles z obarjanjem z etanolom. Urška Rauter, 24. aprila 2015

'''Cepiva''' 

# A novel “priming-boosting” strategy for immune interventions in cervical cancer (S. Liao et al.; Molecular Immunology 64, 295-305, 2015, http://www.sciencedirect.com/science/article/pii/S0161589014003460. Nova "priming-boosting" strategija za imunsko posredovanje pri raku materničnega vratu. Anita Kustec, 8. maja 2015
# Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors (H.C. Wang et al.; Immunology letters 163, 206-213, 2015, http://www.ncbi.nlm.nih.gov/pubmed/25102364 ) Izboljšava cepiv proti antraksu z uporabo iz virusnih replikonov izvedenih vektorjev, ki omogočajo izražanje zaščitnega antigena. Daša Pavc, 8. maja 2015

'''Male molekule in polimeri''' 
Iza Ogris, 15. maja 2015 

# Chromosomal integration of hyaluronic acid synthesis (''has'') genes enhances the molecular weight of hyaluronan produced in ''Lactococcus lactis'' (R. V. Hmar et al; Biotechnol. J. 9 (12), 2014; http://dx.doi.org/10.1002/biot.201400215) Integracija genov za sintezo hialuronske kisline v kromosom bakterije ''Lactococcus lactis'' izboljša sintezo visokomolekularne hialuronske kisline. Maja Grdadolnik, 15. maja 2015

'''Pretvorba biomase''' 
22. maja 2015

'''Metabolično inženirstvo''' 

# Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals (Weerawat Runguphana, Jay D. Keasling; Metabolic Engineering, vol 21, January 2014, Pages 103–113; http://www.sciencedirect.com/science/article/pii/S1096717613000670). Metabolično inženirstvo ''Saccharomyces cerevisiae'' za proizvodnjo derivatov maščobnih kislin, ki so primerni za biogorivo in kemikalije. Dominik Kert, 29. maja 2015

# Metabolic engineering of Klebsiella pneumoniae for the production of cis,cis-muconic acid (Jung,H.-M. Jung,M.-Y. Oh, M.-K.;Applied Microbiology and Biotechnology, Published online: 14 February 2015; http://link.springer.com/article/10.1007/s00253-015-6442-3). Metabolno inženirstvo Klebsiella pneumoniae za produkcijo cis,cis-mukonične kisline. Jure Zabret, 29. maja 2015

'''Biološki viri energije''' 
5. junija 2015

MBT seminarji 2015

2015-03-09T18:01:06Z

MitjaCrcek:

MBT seminarji 2015

2015-03-09T17:53:16Z

MitjaCrcek:

MBT seminarji 2015

2015-03-09T17:51:40Z

MitjaCrcek:

MBT seminarji 2015

2015-03-09T17:48:40Z

MitjaCrcek:

A synthetic multicellular system for programmed pattern formation

2015-01-10T18:05:25Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. ''Figure description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders; see Figure 1a.

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD (band-detect) strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
See [http://www.nature.com./nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

=='''Ring formation dynamics'''==

Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.

For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.

Other experiments show that stability of LacI has the strongest correlation with fluorescence response times and positional shifts. As it was shown ([4]), the stability of LacI affects GFP expression so the LacI decay has an impact on fluorescence. Although dynamics is very important to predict pattern formation, it is not an issue of this topic; for further reading see [4].

=='''Formation of other patterns'''==

The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5] [4]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover.

=='''Summary of pattern formation'''==

Pattern formation is a frequently observed behavior in a prokaryote world. To form a living pattern, cells had to develop communication systems. One of the well described is the AHL signaling system and that is why it was first used to create a synthetic multicellular system for programmed pattern formation.

As we have seen, sender cells use LacI to synthesize acyl-homoserine lactone (AHL). AHL is a small molecule which can diffuse through the cell membrane so it can behave as signaling molecule. Receiver cells uptake the AHL molecule which, in those cells, binds to LuxR. Activated LuxR causes the expression of cI and LacIM1 and those two repressors affect expression of the output protein (e.g. GFP). Sender and receiver cells can be prepared by molecular engineering using different vectors for each type of cells. Those cells are then used to create a pattern on solid media where the driving force for formation is AHL gradient. Time evolution of fluorescence can explain the dynamics of pattern formation, which shows the strongest correlation with stability of LacI. Formation of non-circular patterns is based on the placement and initial concentrations of sender cells and the color of patterns depends on used fluorescent protein.

==References==

[http://www.sciencedirect.com/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.

[http://mmbr.asm.org/content/67/4/574.full.pdf+html] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.

[http://en.wikipedia.org/wiki/Lac_operon] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.

[http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.

[http://link.springer.com/article/10.1007/s10126-001-0081-7] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.

A synthetic multicellular system for programmed pattern formation

2015-01-10T16:58:06Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. ''Figure description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders; see Figure 1a.

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD (band-detect) strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
See [http://www.nature.com./nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

=='''Ring formation dynamics'''==

Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.

For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.

Other experiments show that stability of LacI has the strongest correlation with fluorescence response times and positional shifts. As it was shown ([4]), the stability of LacI affects GFP expression so the LacI decay has an impact on fluorescence. Although dynamics is very important to predict pattern formation, it is not an issue of this topic; for further reading see [4].

=='''Formation of other patterns'''==

The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5] [4]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover.

=='''Summary of pattern formation'''==

Pattern formation is a frequently observed behavior in a prokaryote world. To form a living pattern, cells had to develop communication systems. One of the well described is the AHL signaling system and that is why it was first used to create a synthetic multicellular system for programmed pattern formation.

As we have seen, sender cells use LacI to synthesize acyl-homoserine lactone (AHL). AHL is a small molecule which can diffuse through the cell membrane so it can behave as signaling molecule. Receiver cells uptake the AHL molecule which, in those cells, binds to LuxR. Activated LuxR causes the expression of cI and LacIM1 and those two repressors affect expression of the output protein (e.g. GFP). Sender and receiver cells can be prepared by molecular engineering using different vectors for each type of cells. Those cells are then used to create a pattern on solid media where the driving force for formation is AHL gradient. Time evolution of fluorescence can explain the dynamics of pattern formation, which shows the strongest correlation with stability of LacI. Formation of non-circular patterns is based on the placement and initial concentrations of sender cells and the color of patterns depends on used fluorescent protein.

=='''References'''==

[http://www.sciencedirect.com/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.

[http://mmbr.asm.org/content/67/4/574.full.pdf+html] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.

[http://en.wikipedia.org/wiki/Lac_operon] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.

[http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.

[http://link.springer.com/article/10.1007/s10126-001-0081-7] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.

A synthetic multicellular system for programmed pattern formation

2015-01-10T16:53:07Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. ''Figure description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders; see Figure 1a.

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD (band-detect) strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
See [http://www.nature.com./nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

=='''Ring formation dynamics'''==

Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.

For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.

Other experiments show that stability of LacI has the strongest correlation with fluorescence response times and positional shifts. As it was shown ([4]), the stability of LacI affects GFP expression so the LacI decay has an impact on fluorescence. Although dynamics is very important to predict pattern formation, it is not an issue of this topic; for further reading see [4].

=='''Formation of other patterns'''==

The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover [4].

=='''Summary of pattern formation'''==

Pattern formation is a frequently observed behavior in a prokaryote world. To form a living pattern, cells had to develop communication systems. One of the well described is the AHL signaling system and that is why it was first used to create a synthetic multicellular system for programmed pattern formation.

As we have seen, sender cells use LacI to synthesize acyl-homoserine lactone (AHL). AHL is a small molecule which can diffuse through the cell membrane so it can behave as signaling molecule. Receiver cells uptake the AHL molecule which, in those cells, binds to LuxR. Activated LuxR causes the expression of cI and LacIM1 and those two repressors affect expression of the output protein (e.g. GFP). Sender and receiver cells can be prepared by molecular engineering using different vectors for each type of cells. Those cells are then used to create a pattern on solid media where the driving force for formation is AHL gradient. Time evolution of fluorescence can explain the dynamics of pattern formation, which shows the strongest correlation with stability of LacI. Formation of non-circular patterns is based on the placement and initial concentrations of sender cells and the color of patterns depends on used fluorescent protein.

=='''References'''==

[http://www.sciencedirect.com/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.

[http://mmbr.asm.org/content/67/4/574.full.pdf+html] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.

[http://en.wikipedia.org/wiki/Lac_operon] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.

[http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.

[http://link.springer.com/article/10.1007/s10126-001-0081-7] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.

A synthetic multicellular system for programmed pattern formation

2015-01-10T13:30:31Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. ''Figure description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders; see Figure 1a.

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
See [http://www.nature.com./nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

=='''Ring formation dynamics'''==

Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.

For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.

Other experiments show that stability of LacI has the strongest correlation with fluorescence response times and positional shifts. As it was shown ([4]), the stability of LacI affects GFP expression so the LacI decay has an impact on fluorescence. Although dynamics is very important to predict pattern formation, it is not an issue of this topic; for further reading see [4].

=='''Formation of other patterns'''==

The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover [4].

=='''Summary of pattern formation'''==

Pattern formation is a frequently observed behavior in a prokaryote world. To form a living pattern, cells had to develop communication systems. One of the well described is the AHL signaling system and that is why it was first used to create a synthetic multicellular system for programmed pattern formation.

As we have seen, sender cells use LacI to synthesize acyl-homoserine lactone (AHL). AHL is a small molecule which can diffuse through the cell membrane so it can behave as signaling molecule. Receiver cells uptake the AHL molecule which, in those cells, binds to LuxR. Activated LuxR causes the expression of cI and LacIM1 and those two repressors affect expression of the output protein (e.g. GFP). Sender and receiver cells can be prepared by molecular engineering using different vectors for each type of cells. Those cells are then used to create a pattern on solid media where the driving force for formation is AHL gradient. Time evolution of fluorescence can explain the dynamics of pattern formation, which shows the strongest correlation with stability of LacI. Formation of non-circular patterns is based on the placement and initial concentrations of sender cells and the color of patterns depends on used fluorescent protein.

=='''References'''==

[http://www.sciencedirect.com/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.

[http://mmbr.asm.org/content/67/4/574.full.pdf+html] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.

[http://en.wikipedia.org/wiki/Lac_operon] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.

[http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.

[http://link.springer.com/article/10.1007/s10126-001-0081-7] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.

A synthetic multicellular system for programmed pattern formation

2015-01-10T13:23:02Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon [3]. See figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. ''Figure description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders; see Figure 1a.

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
See [http://www.nature.com./nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

=='''Ring formation dynamics'''==

Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.

For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.

Other experiments show that stability of LacI has the strongest correlation with fluorescence response times and positional shifts. As it was shown ([4]), the stability of LacI affects GFP expression so the LacI decay has an impact on fluorescence. Although dynamics is very important to predict pattern formation, it is not an issue of this topic; for further reading see [4].

=='''Formation of other patterns'''==

The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover [4].

=='''Summary of pattern formation'''==

Pattern formation is a frequently observed behavior in a prokaryote world. To form a living pattern, cells had to develop communication systems. One of the well described is the AHL signaling system and that is why it was first used to create a synthetic multicellular system for programmed pattern formation.

As we have seen, sender cells use LacI to synthesize acyl-homoserine lactone (AHL). AHL is a small molecule which can diffuse through the cell membrane so it can behave as signaling molecule. Receiver cells uptake the AHL molecule which, in those cells, binds to LuxR. Activated LuxR causes the expression of cI and LacIM1 and those two repressors affect expression of the output protein (e.g. GFP). Sender and receiver cells can be prepared by molecular engineering using different vectors for each type of cells. Those cells are then used to create a pattern on solid media where the driving force for formation is AHL gradient. Time evolution of fluorescence can explain the dynamics of pattern formation, which shows the strongest correlation with stability of LacI. Formation of non-circular patterns is based on the placement and initial concentrations of sender cells and the color of patterns depends on used fluorescent protein.

=='''References'''==

[http://www.sciencedirect.com/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.

[http://mmbr.asm.org/content/67/4/574.full.pdf+html] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.

[http://en.wikipedia.org/wiki/Lac_operon] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.

[http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.

[http://link.springer.com/article/10.1007/s10126-001-0081-7] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.

A synthetic multicellular system for programmed pattern formation

2015-01-10T13:20:24Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon [3]. See figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders; see Figure 1a. ''Figure description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
See [http://www.nature.com./nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

=='''Ring formation dynamics'''==

Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.

For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.

Other experiments show that stability of LacI has the strongest correlation with fluorescence response times and positional shifts. As it was shown ([4]), the stability of LacI affects GFP expression so the LacI decay has an impact on fluorescence. Although dynamics is very important to predict pattern formation, it is not an issue of this topic; for further reading see [4].

=='''Formation of other patterns'''==

The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover [4].

=='''Summary of pattern formation'''==

Pattern formation is a frequently observed behavior in a prokaryote world. To form a living pattern, cells had to develop communication systems. One of the well described is the AHL signaling system and that is why it was first used to create a synthetic multicellular system for programmed pattern formation.

As we have seen, sender cells use LacI to synthesize acyl-homoserine lactone (AHL). AHL is a small molecule which can diffuse through the cell membrane so it can behave as signaling molecule. Receiver cells uptake the AHL molecule which, in those cells, binds to LuxR. Activated LuxR causes the expression of cI and LacIM1 and those two repressors affect expression of the output protein (e.g. GFP). Sender and receiver cells can be prepared by molecular engineering using different vectors for each type of cells. Those cells are then used to create a pattern on solid media where the driving force for formation is AHL gradient. Time evolution of fluorescence can explain the dynamics of pattern formation, which shows the strongest correlation with stability of LacI. Formation of non-circular patterns is based on the placement and initial concentrations of sender cells and the color of patterns depends on used fluorescent protein.

=='''References'''==

[http://www.sciencedirect.com/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.

[http://mmbr.asm.org/content/67/4/574.full.pdf+html] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.

[http://en.wikipedia.org/wiki/Lac_operon] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.

[http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.

[http://link.springer.com/article/10.1007/s10126-001-0081-7] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.

A synthetic multicellular system for programmed pattern formation

2015-01-10T13:14:45Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon [3]. See figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders (see Figure 1a).

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

''Figure 1a description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
See [http://www.nature.com./nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

=='''Ring formation dynamics'''==

Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.

For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.

Other experiments show that stability of LacI has the strongest correlation with fluorescence response times and positional shifts. As it was shown ([4]), the stability of LacI affects GFP expression so the LacI decay has an impact on fluorescence. Although dynamics is very important to predict pattern formation, it is not an issue of this topic; for further reading see [4].

=='''Formation of other patterns'''==

The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover [4].

=='''Summary of pattern formation'''==

Pattern formation is a frequently observed behavior in a prokaryote world. To form a living pattern, cells had to develop communication systems. One of the well described is the AHL signaling system and that is why it was first used to create a synthetic multicellular system for programmed pattern formation.

As we have seen, sender cells use LacI to synthesize acyl-homoserine lactone (AHL). AHL is a small molecule which can diffuse through the cell membrane so it can behave as signaling molecule. Receiver cells uptake the AHL molecule which, in those cells, binds to LuxR. Activated LuxR causes the expression of cI and LacIM1 and those two repressors affect expression of the output protein (e.g. GFP). Sender and receiver cells can be prepared by molecular engineering using different vectors for each type of cells. Those cells are then used to create a pattern on solid media where the driving force for formation is AHL gradient. Time evolution of fluorescence can explain the dynamics of pattern formation, which shows the strongest correlation with stability of LacI. Formation of non-circular patterns is based on the placement and initial concentrations of sender cells and the color of patterns depends on used fluorescent protein.

=='''References'''==

[http://www.sciencedirect.com/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.

[http://mmbr.asm.org/content/67/4/574.full.pdf+html] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.

[http://en.wikipedia.org/wiki/Lac_operon] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.

[http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.

[http://link.springer.com/article/10.1007/s10126-001-0081-7] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.

A synthetic multicellular system for programmed pattern formation

2015-01-10T13:03:36Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon [3]. See figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders (see Figure 1a).

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

''Figure 1a description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
See [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

=='''Ring formation dynamics'''==

Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.

For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.

Other experiments show that stability of LacI has the strongest correlation with fluorescence response times and positional shifts. As it was shown ([4]), the stability of LacI affects GFP expression so the LacI decay has an impact on fluorescence. Although dynamics is very important to predict pattern formation, it is not an issue of this topic; for further reading see [4].

=='''Formation of other patterns'''==

The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover [4].

=='''Summary of pattern formation'''==

Pattern formation is a frequently observed behavior in a prokaryote world. To form a living pattern, cells had to develop communication systems. One of the well described is the AHL signaling system and that is why it was first used to create a synthetic multicellular system for programmed pattern formation.

As we have seen, sender cells use LacI to synthesize acyl-homoserine lactone (AHL). AHL is a small molecule which can diffuse through the cell membrane so it can behave as signaling molecule. Receiver cells uptake the AHL molecule which, in those cells, binds to LuxR. Activated LuxR causes the expression of cI and LacIM1 and those two repressors affect expression of the output protein (e.g. GFP). Sender and receiver cells can be prepared by molecular engineering using different vectors for each type of cells. Those cells are then used to create a pattern on solid media where the driving force for formation is AHL gradient. Time evolution of fluorescence can explain the dynamics of pattern formation, which shows the strongest correlation with stability of LacI. Formation of non-circular patterns is based on the placement and initial concentrations of sender cells and the color of patterns depends on used fluorescent protein.

=='''References'''==

[http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.

[http://mmbr.asm.org/content/67/4/574.full.pdf+html] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.

[http://en.wikipedia.org/wiki/Lac_operon] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/full/nature03461.html] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.

[http://link.springer.com.nukweb.nuk.uni-lj.si/article/10.1007/s10126-001-0081-7] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.

A synthetic multicellular system for programmed pattern formation

2015-01-10T13:01:00Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon [3]. See figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders (see Figure 1a).

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

''Figure 1a description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
See [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

=='''Ring formation dynamics'''==

Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.

For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.

Other experiments show that stability of LacI has the strongest correlation with fluorescence response times and positional shifts. As it was shown ([4]), the stability of LacI affects GFP expression so the LacI decay has an impact on fluorescence. Although dynamics is very important to predict pattern formation, it is not an issue of this topic; for further reading see [4].

=='''Formation of other patterns'''==

The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover [4].

=='''Summary of pattern formation'''==

Pattern formation is a frequently observed behavior in a prokaryote world. To form a living pattern, cells had to develop communication systems. One of the well described is the AHL signaling system and that is why it was first used to create a synthetic multicellular system for programmed pattern formation.

As we have seen, sender cells use LacI to synthesize acyl-homoserine lactone (AHL). AHL is a small molecule which can diffuse through the cell membrane so it can behave as signaling molecule. Receiver cells uptake the AHL molecule which, in those cells, binds to LuxR. Activated LuxR causes the expression of cI and LacIM1 and those two repressors affect expression of the output protein (e.g. GFP). Sender and receiver cells can be prepared by molecular engineering using different vectors for each type of cells. Those cells are then used to create a pattern on solid media where the driving force for formation is AHL gradient. Time evolution of fluorescence can explain the dynamics of pattern formation, which shows the strongest correlation with stability of LacI. Formation of non-circular patterns is based on the placement and initial concentrations of sender cells and the color of patterns depends on used fluorescent protein.

=='''References'''==

[http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.

[http://mmbr.asm.org/content/67/4/574.full.pdf+html] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.

[http://en.wikipedia.org/wiki/Lac_operon] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/full/nature03461.html] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.

[http://link.springer.com.nukweb.nuk.uni-lj.si/article/10.1007/s10126-001-0081-7] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.

A synthetic multicellular system for programmed pattern formation

2015-01-10T12:50:46Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon [3]. See figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders (see Figure 1a).

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

''Figure 1a description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
See [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

=='''Ring formation dynamics'''==

Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.

For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.

Other experiments show that stability of LacI has the strongest correlation with fluorescence response times and positional shifts. As it was shown ([4]), the stability of LacI affects GFP expression so the LacI decay has an impact on fluorescence. Although dynamics is very important to predict pattern formation, it is not an issue of this topic; for further reading see [4].

=='''Formation of other patterns'''==

The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover [4].

=='''Summary of pattern formation'''==

Pattern formation is a frequently observed behavior in a prokaryote world. To form a living pattern, cells had to develop communication systems. One of the well described is the AHL signaling system and that is why it was first used to create a synthetic multicellular system for programmed pattern formation.

As we have seen, sender cells use LacI to synthesize acyl-homoserine lactone (AHL). AHL is a small molecule which can diffuse through the cell membrane so it can behave as signaling molecule. Receiver cells uptake the AHL molecule which, in those cells, binds to LuxR. Activated LuxR causes the expression of cI and LacIM1 and those two repressors affect expression of the output protein (e.g. GFP). Sender and receiver cells can be prepared by molecular engineering using different vectors for each type of cells. Those cells are then used to create a pattern on solid media where the driving force for formation is AHL gradient. Time evolution of fluorescence can explain the dynamics of pattern formation, which shows the strongest correlation with stability of LacI. Formation of non-circular patterns is based on the placement and initial concentrations of sender cells and the color of patterns depends on used fluorescent protein.

=='''References'''==

[http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.

[http://mmbr.asm.org/content/67/4/574.full.pdf+html] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.

[http://en.wikipedia.org/wiki/Lac_operon] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/full/nature03461.html] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.

[http://link.springer.com.nukweb.nuk.uni-lj.si/article/10.1007/s10126-001-0081-7] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.

A synthetic multicellular system for programmed pattern formation

2015-01-10T12:48:01Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon [3]. See figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders (see Figure 1a).

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

''Figure 1a description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
See [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

=='''Ring formation dynamics'''==

Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.

For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.

Other experiments show that stability of LacI has the strongest correlation with fluorescence response times and positional shifts. As it was shown ([4]), the stability of LacI affects GFP expression so the LacI decay has an impact on fluorescence. Although dynamics is very important to predict pattern formation, it is not an issue of this topic; for further reading see [4].

=='''Formation of other patterns'''==

The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover [4].

=='''Summary of pattern formation'''==

Pattern formation is a frequently observed behavior in a prokaryote world. To form a living pattern, cells had to develop communication systems. One of the well described is the AHL signaling system and that is why it was first used to create a synthetic multicellular system for programmed pattern formation.

As we have seen, sender cells use LacI to synthesize acyl-homoserine lactone (AHL). AHL is a small molecule which can diffuse through the cell membrane so it can behave as signaling molecule. Receiver cells uptake the AHL molecule which, in those cells, binds to LuxR. Activated LuxR causes the expression of cI and LacIM1 and those two repressors affect expression of the output protein (e.g. GFP). Sender and receiver cells can be prepared by molecular engineering using different vectors for each type of cells. Those cells are then used to create a pattern on solid media where the driving force for formation is AHL gradient. Time evolution of fluorescence can explain the dynamics of pattern formation, which shows the strongest correlation with stability of LacI. Formation of non-circular patterns is based on the placement and initial concentrations of sender cells and the color of patterns depends on used fluorescent protein.

=='''References'''==

[http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.

[http://mmbr.asm.org/content/67/4/574.full.pdf+html] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.

[http://en.wikipedia.org/wiki/Lac_operon] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/full/nature03461.html] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.

[http://download.springer.com.nukweb.nuk.uni-lj.si/static/pdf/256/art%253A10.1007%252Fs10126-001-0081-7.pdf?auth66=1420894086_cacdf94c6c231719ca3ef3a53b6096e8&ext=.pdf] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.

A synthetic multicellular system for programmed pattern formation

2015-01-10T12:45:24Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon [3]. See figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders (see Figure 1a).

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

''Figure 1a description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
See [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

=='''Ring formation dynamics'''==

Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.

For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.

Other experiments show that stability of LacI has the strongest correlation with fluorescence response times and positional shifts. As it was shown ([4]), the stability of LacI affects GFP expression so the LacI decay has an impact on fluorescence. Although dynamics is very important to predict pattern formation, it is not an issue of this topic; for further reading see [4].

=='''Formation of other patterns'''==

The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover [4].

=='''Summary of pattern formation'''==

Pattern formation is a frequently observed behavior in a prokaryote world. To form a living pattern, cells had to develop communication systems. One of the well described is the AHL signaling system and that is why it was first used to create a synthetic multicellular system for programmed pattern formation.

As we have seen, sender cells use LacI to synthesize acyl-homoserine lactone (AHL). AHL is a small molecule which can diffuse through the cell membrane so it can behave as signaling molecule. Receiver cells uptake the AHL molecule which, in those cells, binds to LuxR. Activated LuxR causes the expression of cI and LacIM1 and those two repressors affect expression of the output protein (e.g. GFP). Sender and receiver cells can be prepared by molecular engineering using different vectors for each type of cells. Those cells are then used to create a pattern on solid media where the driving force for formation is AHL gradient. Time evolution of fluorescence can explain the dynamics of pattern formation, which shows the strongest correlation with stability of LacI. Formation of non-circular patterns is based on the placement and initial concentrations of sender cells and the color of patterns depends on used fluorescent protein.

=='''References'''==

[http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.

[http://mmbr.asm.org/content/67/4/574.full.pdf+html] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.

[3] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.

[4] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.

[5] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.

A synthetic multicellular system for programmed pattern formation

2015-01-10T12:44:35Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon [3]. See figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders (see Figure 1a).

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

''Figure 1a description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
See [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

=='''Ring formation dynamics'''==

Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.

For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.

Other experiments show that stability of LacI has the strongest correlation with fluorescence response times and positional shifts. As it was shown ([4]), the stability of LacI affects GFP expression so the LacI decay has an impact on fluorescence. Although dynamics is very important to predict pattern formation, it is not an issue of this topic; for further reading see [4].

=='''Formation of other patterns'''==

The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover [4].

=='''Summary of pattern formation'''==

Pattern formation is a frequently observed behavior in a prokaryote world. To form a living pattern, cells had to develop communication systems. One of the well described is the AHL signaling system and that is why it was first used to create a synthetic multicellular system for programmed pattern formation.

As we have seen, sender cells use LacI to synthesize acyl-homoserine lactone (AHL). AHL is a small molecule which can diffuse through the cell membrane so it can behave as signaling molecule. Receiver cells uptake the AHL molecule which, in those cells, binds to LuxR. Activated LuxR causes the expression of cI and LacIM1 and those two repressors affect expression of the output protein (e.g. GFP). Sender and receiver cells can be prepared by molecular engineering using different vectors for each type of cells. Those cells are then used to create a pattern on solid media where the driving force for formation is AHL gradient. Time evolution of fluorescence can explain the dynamics of pattern formation, which shows the strongest correlation with stability of LacI. Formation of non-circular patterns is based on the placement and initial concentrations of sender cells and the color of patterns depends on used fluorescent protein.

=='''References'''==

[http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.

[2] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.

[3] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.

[4] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.

[5] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.

A synthetic multicellular system for programmed pattern formation

2015-01-10T12:43:19Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon [3]. See figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders (see Figure 1a).

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

''Figure 1a description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
See [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

=='''Ring formation dynamics'''==

Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.

For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.

Other experiments show that stability of LacI has the strongest correlation with fluorescence response times and positional shifts. As it was shown ([4]), the stability of LacI affects GFP expression so the LacI decay has an impact on fluorescence. Although dynamics is very important to predict pattern formation, it is not an issue of this topic; for further reading see [4].

=='''Formation of other patterns'''==

The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover [4].

=='''Summary of pattern formation'''==

Pattern formation is a frequently observed behavior in a prokaryote world. To form a living pattern, cells had to develop communication systems. One of the well described is the AHL signaling system and that is why it was first used to create a synthetic multicellular system for programmed pattern formation.

As we have seen, sender cells use LacI to synthesize acyl-homoserine lactone (AHL). AHL is a small molecule which can diffuse through the cell membrane so it can behave as signaling molecule. Receiver cells uptake the AHL molecule which, in those cells, binds to LuxR. Activated LuxR causes the expression of cI and LacIM1 and those two repressors affect expression of the output protein (e.g. GFP). Sender and receiver cells can be prepared by molecular engineering using different vectors for each type of cells. Those cells are then used to create a pattern on solid media where the driving force for formation is AHL gradient. Time evolution of fluorescence can explain the dynamics of pattern formation, which shows the strongest correlation with stability of LacI. Formation of non-circular patterns is based on the placement and initial concentrations of sender cells and the color of patterns depends on used fluorescent protein.

=='''References'''==

[1][http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.

[2] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.

[3] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.

[4] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.

[5] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.

A synthetic multicellular system for programmed pattern formation

2015-01-10T12:40:29Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon [3]. See figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders (see Figure 1a).

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

''Figure 1a description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
See [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

=='''Ring formation dynamics'''==

Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.

For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.

Other experiments show that stability of LacI has the strongest correlation with fluorescence response times and positional shifts. As it was shown ([4]), the stability of LacI affects GFP expression so the LacI decay has an impact on fluorescence. Although dynamics is very important to predict pattern formation, it is not an issue of this topic; for further reading see [4].

=='''Formation of other patterns'''==

The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover [4].

=='''Summary of pattern formation'''==

Pattern formation is a frequently observed behavior in a prokaryote world. To form a living pattern, cells had to develop communication systems. One of the well described is the AHL signaling system and that is why it was first used to create a synthetic multicellular system for programmed pattern formation.

As we have seen, sender cells use LacI to synthesize acyl-homoserine lactone (AHL). AHL is a small molecule which can diffuse through the cell membrane so it can behave as signaling molecule. Receiver cells uptake the AHL molecule which, in those cells, binds to LuxR. Activated LuxR causes the expression of cI and LacIM1 and those two repressors affect expression of the output protein (e.g. GFP). Sender and receiver cells can be prepared by molecular engineering using different vectors for each type of cells. Those cells are then used to create a pattern on solid media where the driving force for formation is AHL gradient. Time evolution of fluorescence can explain the dynamics of pattern formation, which shows the strongest correlation with stability of LacI. Formation of non-circular patterns is based on the placement and initial concentrations of sender cells and the color of patterns depends on used fluorescent protein.

=='''References'''==

[http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S1084952102000186 [1]] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.
[2] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.
[3] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.
[4] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.
[5] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.

A synthetic multicellular system for programmed pattern formation

2015-01-10T12:36:14Z

MitjaCrcek:

A synthetic multicellular system for programmed pattern formation

2015-01-10T12:28:35Z

MitjaCrcek:

A synthetic multicellular system for programmed pattern formation

2015-01-10T12:18:48Z

MitjaCrcek:

A synthetic multicellular system for programmed pattern formation

2015-01-10T12:16:31Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon [3]. See figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders (see Figure 1a).

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

''Figure 1a description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
See [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

Experiments shows the formation of such pattern (see Figure 3b and 3c) on a plate with BD3 or BD1 cells (with gfp gene) and BD2-Red cells (similar to BD2 with dsRed-Express replacing gfp gene). First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

A synthetic multicellular system for programmed pattern formation

2015-01-10T12:14:53Z

MitjaCrcek:

('''Mitja Crček''')

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

Nature 434, 1130-1134 (28 April 2005)

'''INTRODUCTION'''

Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.

Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, ''lux'' operon, ''lac'' operon and lac repressor to explain the molecular basis and show the role of AHL.

In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.

In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.

=='''AHL signaling'''==

Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, ''lux'' operon, formation of AHL and the lac repressor.

===Tet regulatory system===

Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).

There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].

===''Lux'' operon===

The ''lux'' operon encodes genes for self-regulation and was first found in ''Vibrio fischeri''. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 [http://mmbr.asm.org/content/67/4/574.full.pdf+html here] [2]. ''Figure description'': Quorum-sensing model in ''P. fischeri'' using ''lux'' operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene ''lux''R is under the constitutive promoter). Active LuxR can bind to ''pLux''R promoter which leads to transcription of other genes in ''lux'' operon, also ''lux''I (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].

''Lux''R gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.

LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].

With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.

Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.

===''Lac'' operon and Lac repressor===

The ''lac'' operon or lactose operon is an operon with the task for transport and metabolism of lactose in E.coli and some other enteric bacteria. It has three structural genes, ''lac''Z, ''lac''Y and ''lac''A which encode β-galactosidase, lactose permease, and galactoside O-acetyltransferase, respectively; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_operon1.png here] [3]. The lac operon uses a well-regulated control mechanism so that ''lac'' genes are expressed only when necessary (i.e. at high concentration of lactose or analogs). Transcription of the ''lac'' operon genes is suppressed when LacI repressor coded by ''lac''I gene binds to lac operator.

The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon [3]. See figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

=='''Signaling process and output protein production'''==

[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders (see Figure 1a).

AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

Receiver cells that are close to the sender cells receive high concentrations of AHL, resulting in high cytoplasmic levels of LacIM1 and cI. Although cI disables the expression of LacI, the level of LacIM1 is high enough to repress the expression of GFP, resulting in absence of the mentioned protein. Receivers that are far from the sender cells have very low concentration of AHL or no AHL at all, resulting in inactive protein LuxR. That means there is no expression of LacIM1 and also cI, which is unable to repress the expression of wild type LacI, resulting again in GFP repression. In both cases we cannot detect the fluorescence of the cells (see Figure 1a).

At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

''Figure 1a description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

=='''Pattern formation'''==

===Plasmids for sender and receiver cells===

To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

High-detect plasmid (pHD{x} - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
Low-detect plasmid (pLD - see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].

===Simulated and experimental behavior of engineered bacterial strains===

Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

===Circular pattern formation===

Spatiotemporal simulations of a system predicted that by placing sender cells next to receiver cells, the described network could direct pattern formation on solid media. If we place sender cells in the middle of the plate, a circular pattern is expected to be formed. Furthermore, a bullseye pattern could be achieved by mixing different variants such as BD1, BD2 and/or BD3.

Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with gfp gene) and BD2-Red cells (similar to BD2 with dsRed-Express replacing gfp gene).
See [http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

Experiments shows the formation of such pattern (see Figure 3b and 3c) on a plate with BD3 or BD1 cells (with gfp gene) and BD2-Red cells (similar to BD2 with dsRed-Express replacing gfp gene). First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish Aequorea macrodactyla [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).

Similarly, when BD2-Red cells and BD1 cells were mixed and plated with a sender disk, green fluorescent disk was detected around the red fluorescent ring (see Figure 3c). Different diameters of the two BD2-Red rings (30 mm versus 22 mm) can be explained by variations of the AHL gradients due to differences in population of receivers and senders as well as in the growth rates. Subtle environmental differences between experiments such as agar densities and nutrient conditions can also affect different ring formation.

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