Wiki FKKT - User contributions [en]

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T18:04:43Z

Alminatahirovic:

Edited according to the article: [https://link.springer.com/article/10.1007/s12033-020-00243-1 Saeed, S., Mehreen, H., Gerlevik, U., Tariq, A., Manzoor, S., Noreen et Sezerman, U: HriGFP Novel Flourescent Protein: Expression and Applications. Molecular Biotechnology. 2020, 62: 280-288]

Author: Almina Tahirović

==Introduction==
Enviromental pollution is one of the greatest global issues to this day and it keeps increasing at an alarming level which poses a great danger to human health. The greatest threats are in fact, industrial wastes and pollutants which are constantly released into the enviroment, including the pesticides that consist of organo-phosphorous compounds or either their derivatives [1]. Those pesticided are often used to increase the food production, even though the high amounts of it can have lethal consequences when it comes to human health or food safety. Since the proper remoting of agricultural areas have been problem for decades, because the usual methods like Chromatography Mass Spectroscopy or Liquid/Gas Chromatography are costly, non-portable and require specialized laboratories, scientist have come up with the idea of using cost-effective portable biosensors which could effectively detect the methyl parathion (one of the organo-phosphorous compounds) on spot [2].

==Green fluorescent protein==
The green fluorescent protein (GFP) is a protein that sucessfully exhibits green fluorescence when exposed to light in the blue to ultraviolet range and GFP usually refers to the protein first isolated from the jellyfish ''Aequorea victoria''. Also, GFP-like coral proteins exhibit a broad spectral diversity ranging from blue to red FPs, so both the stability and expression of it could be optimized by amino acids substitutions possessing different kinds of biophysical properties, therefore such modifications could ensure better rate of maturation and stability of mentioned FPs. Such modificated and more stable monomeric FPs could be used as biosensors [3].

==Matherials and methods==
Materials used in this study were of analytical grade - stock and arsenium solution, both obtained from Sigma. HriGFP gene was cloned into pET28a+vector (containing the T7 lac promoter, induced using isopropyl β-d-1-thiogalactopyranoside) and sucessfully transformed into ''E. coli'' BL21 (DE3) cells. In order to sucessfully transform it in ''Bacillus megaterium'', the gene had to be sub-cloned into the shuttle vector pHIS1522. Kanamycin was added as well, with the role of the resistant marker and the plates were incubated at 37 °C overnight. Structure prediction of the HriGFP was done by homology modeling and entire statistical analysis was performed with Graph-pad Prism. Lastly, all the biosensing experiments were done in total of three biological replicates [1].

==Results==
Research team sucessfully transformed and expressed the HriGFP in ''E. coli'' and ''Bacillus megaterium'' which was presented by fluorescent microscopy. The entire HriGFP structure was modeled with the help of MODELLER - it contained 7 beta sheets, resembling to beta barrel which is specific for GFP molecules. Research team also analyzed the HriGFP interaction with the metal ions where strong interaction was observed with divalent iones, such as Mg and some slightly less strong interactions with iones like Zn and Mn. It was also observed that exposure of HriGFP to the monovalent iones did not show any change in fuorescence intensity and at final, it was concluded that strongest fuorescence quenching was observed in the case of copper as opposed to mercury and arsenic. Alltoghether, the biosensing ability of the HriGFP producing cells was tested and signifcant increase in the fuorescence intensity was observed with the
corresponding decrease in the concentration of methyl parathion, which is considered to be detected from 500 mg to 0.05 mg/L (1.89 mM- 189 μM) concentration [1].

==Conclusion==
HriGFP could be used efciently as biosensor for the detection of heavy metals and harmful organo-phosphorous compounds like methyl-parahtion.

==Literature==

[1] Saeed, S., Mehreen, H., Gerlevik, U., Tariq, A., Manzoor, S., Noreen, Z., Sezerman, U. HriGFP Novel Flourescent Protein: Expression and Applications. ''Molecular Biotechnology''. '''2020''';62: 280-288.

[2] Gong, J., Wang, L., Zhang, L. Electrochemical biosensing of methyl parathion pesticide based on acetylcholinesterase
immobilized onto Au–polypyrrole interlaced network-like nano-composite. ''Biosensors and Bioelectronics''. '''2009''';24: 2285–2288.

[3] Mérola, F., Erard, M., Fredj, A., Pasquier, H. Engineering fuorescent proteins towards ultimate performances: lessons
from the newly developed cyan variants.'' Methods and Applications in Fluorescence''. '''2016''';4: 1200.

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T18:04:11Z

Alminatahirovic:

Edited according to the article: [https://link.springer.com/article/10.1007/s12033-020-00243-1 Saeed, S., Mehreen, H., Gerlevik, U., Tariq, A., Manzoor, S., Noreen et Sezerman, U: HriGFP Novel Flourescent Protein: Expression and Applications. Molecular Biotechnology. 2020, 62: 280-288]

==Introduction==
Enviromental pollution is one of the greatest global issues to this day and it keeps increasing at an alarming level which poses a great danger to human health. The greatest threats are in fact, industrial wastes and pollutants which are constantly released into the enviroment, including the pesticides that consist of organo-phosphorous compounds or either their derivatives [1]. Those pesticided are often used to increase the food production, even though the high amounts of it can have lethal consequences when it comes to human health or food safety. Since the proper remoting of agricultural areas have been problem for decades, because the usual methods like Chromatography Mass Spectroscopy or Liquid/Gas Chromatography are costly, non-portable and require specialized laboratories, scientist have come up with the idea of using cost-effective portable biosensors which could effectively detect the methyl parathion (one of the organo-phosphorous compounds) on spot [2].

==Green fluorescent protein==
The green fluorescent protein (GFP) is a protein that sucessfully exhibits green fluorescence when exposed to light in the blue to ultraviolet range and GFP usually refers to the protein first isolated from the jellyfish ''Aequorea victoria''. Also, GFP-like coral proteins exhibit a broad spectral diversity ranging from blue to red FPs, so both the stability and expression of it could be optimized by amino acids substitutions possessing different kinds of biophysical properties, therefore such modifications could ensure better rate of maturation and stability of mentioned FPs. Such modificated and more stable monomeric FPs could be used as biosensors [3].

==Matherials and methods==
Materials used in this study were of analytical grade - stock and arsenium solution, both obtained from Sigma. HriGFP gene was cloned into pET28a+vector (containing the T7 lac promoter, induced using isopropyl β-d-1-thiogalactopyranoside) and sucessfully transformed into ''E. coli'' BL21 (DE3) cells. In order to sucessfully transform it in ''Bacillus megaterium'', the gene had to be sub-cloned into the shuttle vector pHIS1522. Kanamycin was added as well, with the role of the resistant marker and the plates were incubated at 37 °C overnight. Structure prediction of the HriGFP was done by homology modeling and entire statistical analysis was performed with Graph-pad Prism. Lastly, all the biosensing experiments were done in total of three biological replicates [1].

==Results==
Research team sucessfully transformed and expressed the HriGFP in ''E. coli'' and ''Bacillus megaterium'' which was presented by fluorescent microscopy. The entire HriGFP structure was modeled with the help of MODELLER - it contained 7 beta sheets, resembling to beta barrel which is specific for GFP molecules. Research team also analyzed the HriGFP interaction with the metal ions where strong interaction was observed with divalent iones, such as Mg and some slightly less strong interactions with iones like Zn and Mn. It was also observed that exposure of HriGFP to the monovalent iones did not show any change in fuorescence intensity and at final, it was concluded that strongest fuorescence quenching was observed in the case of copper as opposed to mercury and arsenic. Alltoghether, the biosensing ability of the HriGFP producing cells was tested and signifcant increase in the fuorescence intensity was observed with the
corresponding decrease in the concentration of methyl parathion, which is considered to be detected from 500 mg to 0.05 mg/L (1.89 mM- 189 μM) concentration [1].

==Conclusion==
HriGFP could be used efciently as biosensor for the detection of heavy metals and harmful organo-phosphorous compounds like methyl-parahtion.

==Literature==

[1] Saeed, S., Mehreen, H., Gerlevik, U., Tariq, A., Manzoor, S., Noreen, Z., Sezerman, U. HriGFP Novel Flourescent Protein: Expression and Applications. ''Molecular Biotechnology''. '''2020''';62: 280-288.

[2] Gong, J., Wang, L., Zhang, L. Electrochemical biosensing of methyl parathion pesticide based on acetylcholinesterase
immobilized onto Au–polypyrrole interlaced network-like nano-composite. ''Biosensors and Bioelectronics''. '''2009''';24: 2285–2288.

[3] Mérola, F., Erard, M., Fredj, A., Pasquier, H. Engineering fuorescent proteins towards ultimate performances: lessons
from the newly developed cyan variants.'' Methods and Applications in Fluorescence''. '''2016''';4: 1200.

MBT seminarji 2021

2021-05-24T17:55:47Z

Alminatahirovic:

Seminarji iz Molekularne biotehnologije so letos organizirani tako, da vsak študent (praviloma v paru, lahko pa tudi samostojno) obdela temo s področja cepiv proti virusu SARS-CoV-2 in o tem pripravi kratek poljudno napisan povzetek. Ta del seminarjev je predstavljen na [[protikovidna cepiva|ločeni strani]].
V drugem delu vsak študent predstavi nek raziskovalni dosežek s širšega področja molekularne biotehnologije. Seznam tem in predstavitev za študijsko leto 2020/21 je predstavljen tu.

Povzetke morate objaviti do torka do polnoči v tednu, ko imate seminar (v četrtek). 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. predlani).

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 (28.2.) 
(slovenski naslov povežite z novo stranjo, na kateri bo povzetek)

----

Naslovi odobrenih člankov po temah:

'''Farmacevtsko pomembni proteini''' 
# Development of Antibody-Fragment-Producing Rice for Neutralization of Human Norovirus (A. Sasou ''et. al''; Frontiers in Plant Science 12, 2021; https://doi.org/10.3389/fpls.2021.639953). [[Proizvodnja riža za sintezo fragmentov protiteles proti humanemu norovirusu.]] Mateja Žvipelj (11.3.)
# A New Plant Expression System for Producing Pharmaceutical Proteins (N. Abd-Aziz ''et. al''; Molecular Biotechnology 62, 2020; https://doi.org/10.1007/s12033-020-00242-2). [[Razvoj ekspresijskega sistema za proizvodnjo farmacevtskih proteinov v rastlini Mucuna bracteata]]. Jernej Imperl (18.3.)
# Development of a Recombinant Monospecific Anti-PLGF Bivalent Nanobody and Evaluation of it in Angiogenesis Modulation (A. Nikooharf "et all"; Molecular Biotechnology 62, 2020; https://link.springer.com/article/10.1007/s12033-020-00275-7#additional-information) [[Razvoj rekombinantnih monospecifičnih bivalentnih nanoteles proti PLGF-u]]. Nika Zaveršek (18.3.)

'''Cepiva''' 
# Development of a DNA Vaccine for Melanoma Metastasis by Inhalation Based on an Analysis of Transgene Expression Characteristics of Naked pDNA and a Ternary Complex in Mouse Lung Tissues (Kodama ''et.al'';Pharmaceutics 12,2020; https://www.mdpi.com/1999-4923/12/6/540#framed_div_cited_count) [[ Razvoj DNA cepiva proti metastazam melanoma z vdihavanjem na podlagi analize značilnosti transgene ekspresije gole pDNA in trojni kompleks v mišjem pljučnem tkivu]]. Paula Horvat (25.3.) 
# An AMA1/MSP119 Adjuvanted Malaria Transplastomic Plant‑Based Vaccine Induces Immune Responses in Test Animals (Evelia M. Milán‑Noris ''et. al''; Molecular Biotechnology 62, 2020; https://doi.org/10.1007/s12033-020-00271-x) [[V rastlinah proizvedeno transplastomsko antimalarijsko cepivo z AMA1/MSP119 in dodanim adjuvansom inducira imunski odziv v testnih živalih]]. Neža Pavko (25.3.) 

'''Gensko spremenjene rastline''' 
# A wheat cysteine-rich receptor-like kinase confers broad-spectrum resistance against Septoria tritici blotch (C. Saintenac ''et al.''; Nat. Commun. 12, 2021, https://doi.org/10.1038/s41467-020-20685-0). [[Receptorju podobna kinaza bogata s cisteini, pšenici daje odpornost proti širokemu spektru pegavosti Septoria tritici]]. Andrej Race (7.4.)
# RNAi silenced ζ-carotene desaturase developed variegated tomato transformants with increased phytoene content (M. A. Babu ''et. al''; Plant Growth Regul. 93, 2021; https://doi.org/10.1007/s10725-020-00678-1). [[Vpliv utišanja ζ-karoten desaturaze na vsebnost karotenoidov v gensko spremenjenih paradižnikih]]. Peter Škrinjar (7.4.)

'''Gensko spremenjene živali in celične linije''' 
# Engineering carotenoid production in mammalian cells for nutritionally enhanced cell-cultured foods (A. J. Stout "et. al"; Metabolic Engineering 62, 2020; https://doi.org/10.1016/j.ymben.2020.07.011). [[Razvoj proizvodnje karotenoidov v sesalskih celicah za prehransko izboljšano celično pridobljeno meso]]. Urša Lovše (8.4.)
# Efficient photoactivatable Dre recombinase for cell type-specific spatiotemporal control of genome engineering in the mouse (H. Li ''et. al''; Proc. Natl. Acad. Sci. U. S. A. 117(52), 2021; https://doi.org/10.1073/pnas.2003991117). [[Priprava fotoinducibilne rekombinaze Dre kot orodje za prostorsko in časovno odvisno urejanje genoma v specifičnih mišjih celicah.]] Matija Ruparčič (8.4.)

'''Nizkomolekularni biotehnološki produkti''' 
# Fermentative N-Methylanthranilate Production by Engineered ''Corynebacterium glutamicum''. (T. Walter ''et. al.''; Microorganisms 8(6), 2020; https://doi.org/10.3390/microorganisms8060866). [[Fermentativna proizvodnja N-metilantranilata z inženirsko spremenjeno Corynebacterium glutamicum]]. Saša Slabe (14.4.)
# Efficient Biosynthesis of Vanillin from Isoeugenol by Recombinant Isoeugenol Monooxygenase from ''Pseudomonas nitroreducens'' Jin1. (Wang Q, Wu X, Lu X, He Y, Ma B, Xu Y. Efficient Biosynthesis of Vanillin from Isoeugenol by Recombinant Isoeugenol Monooxygenase from Pseudomonas nitroreducens Jin1. Appl Biochem Biotechnol. 2021:1116-1128. doi:10.1007/s12010-020-03478-5). [[Učinkovita biosinteza vanilina iz izoevgenola z uporabo rekombinantne izoevgenol monooksigenaze Jin1 iz bakterije Pseudomonas nitroreducens]]. Luka Gnidovec (15.4.)
# One-pot production of butyl butyrate from glucose using a cognate “diamond-shaped” ''E. coli'' consortium (J. P. Sinumvayo "et. al"; Bioresources and Bioprocessing 8, 2021; https://bioresourcesbioprocessing.springeropen.com/articles/10.1186/s40643-021-00372-8#Sec9). [[Proizvodnja butil butirata iz glukoze z uporabo "diamantnega" konzorcija E. coli]] Liza Ulčakar (15.4.)

'''Biotehnološki polimeri''' 
# Biotechnologically produced fucosylated oligosaccharides inhibit the binding of human noroviruses to their natural receptors (S. M. Derya et al., “Biotechnologically produced fucosylated oligosaccharides inhibit the binding of human noroviruses to their natural receptors,” ''J. Biotechnol.'', vol. 318, no. April, pp. 31–38, 2020, doi: 10.1016/j.jbiotec.2020.05.001). [[Inhibicija vezave humanega norovirusa na naravni receptor z biotehnološko proizvedenimi fukoziliranimi oligosaharidi]] Anže Karlek (21.4.)
# Complete biosynthesis of a sulfated chondroitin in ''Escherichia coli'' (Badri, A., ''et al''; Nature communications 12 (2021); https://doi.org/10.1038/s41467-021-21692-5). [[Popolna biosinteza hondroitin sulfata v E. coli]] Ana Maklin (22.4.)
# Optimization of cultivation medium and cyclic fed-batch fermentation strategy for enhanced polyhydroxyalkanoate production by Bacillus thuringiensis using a glucose-rich hydrolyzate (Singh et al. Bioresour. Bioprocess. (2021) 8:11, https://doi.org/10.1186/s40643-021-00361-x) [[Optimizacija fermentacijske proizvodnje PHA-bioplastike z b. thuringiensis in z glukozo bogatimi hidrolizati]] Urban Hribar (22.4.)

'''Biotehnološko pridobljeni encimi''' 
# Engineering a carboxypeptidase from ''Aspergillus niger'' M00988 by mutation to increase its ability in high Fischer ratio oligopeptide preparation (Xiong K., Liu J., Wang X., Sun B., Zhang Y., Zhao Z., Pei P., & Li X.; Journal of Biotechnology, 330, 1–8, 2021, https://doi.org/10.1016/j.jbiotec.2021.02.015). [[Priprava karboksipeptidaze iz glive Aspergillus niger M00988 za izboljšanje priprave oligopeptidov z visokim Fischerjevim razmerjem]] Urška Fajdiga (5.5.)
# Cell-Based High-Throughput Screening Protocol for Discovering Antiviral Inhibitors Against SARS-COV-2 Main Protease (3CLpro) (Rothan, H.A., Teoh, T.C; Mol Biotechnol 63, 240–248 (2021); https://doi.org/10.1007/s12033-021-00299-7) [[Visoko zmogljiv presejalni protokol na osnovi celic za raziskovanje antivirusnih inhibitorjev proti Sars-Cov-2 glavni proteazi (3CLpro)]] Mirsad Mešić (6.5.)
# A novel cold-active type I pullulanase from a hot-spring metagenome for effective debranching and production of resistant starch (M. Thakur ''et al''.; Bioresource Technology 320, 2021; https://doi.org/10.1016/j.biortech.2020.124288). [[Pri nizkih temperaturah aktivna pululanaza tipa I iz metagenoma vročih vrelcev omogoča učinkovito klestenje in proizvodnjo odpornega škroba]] Martina Lokar (6.5.)

'''Metabolno inženirstvo v biotehnologiji''' 
# Production of Tyrian purple indigoid dye from tryptophan in ''Escherichia coli'' (J. Lee ''et al.''; Nat. Chem. Biol. 17, 2021; https://doi.org/10.1038/s41589-020-00684-4). [http://wiki.fkkt.uni-lj.si/index.php/Proizvodnja_%C5%A1krlatnega_indigoidnega_barvila_iz_triptofana_v_bakteriji_Escherichia_coli Proizvodnja škrlatnega indigoidnega barvila iz triptofana v bakteriji ''Escherichia coli''] Jerneja Nimac (12.5.)
# Development of ''Pseudomonas asiatica'' as a host for the production of 3-hydroxypropionic acid from glycerol (T. Thi Nguyen et al., Bioresource Technology, vol. 329, 2021; https://doi.org/10.1016/j.biortech.2021.124867). [[Razvoj gostiteljskega organizma Pseudomonas asiatica za proizvodnjo 3-hidroksipropionske kisline iz glicerola]] Urška Pečarič Strnad (12.5.)
# Generation of an engineered food-grade ''Lactococcus lactis'' strain for production of an antimicrobial peptide: ''in vitro'' and ''in silico'' evaluation (A. Tanhaeian ''et. al''; BMC Biotechnol. 20(1), 2020; https://doi.org/10.1186/s12896-020-00612-3). [[Priprava in ovrednotenje novega seva bakterij Lactococcus lactis za proizvodnjo protimikrobnega peptida]]. Klementina Polanec (13.5.)
# Metabolic engineering of ''E. coli'' for producing phloroglucinol from acetate (S. Yu et. al; Applied Microbiology and Biotechnology. 2020; https://doi.org/10.1007/s00253-020-10591-2). [[Metabolno inženirstvo bakterije Escherichia coli za pridobivanje floroglucinola iz acetata]]. Ernestina Lavrih (13.5.)

'''Biomasa in biogoriva''' 
# Green Deep Eutectic Solvents for ''Microwave-Assisted Biomass'' Delignification and Valorisation (Grillo G. ''et al.''; Molecules 2021; https://www.mdpi.com/1420-3049/26/4/798/htm) [[Zelena globoka evtektična topila za delignifikacijo in valorizacijo biomase s pomočjo mikrovalov]]. Željka Erić (19.5.)
# Incorporating a molecular antenna in diatom microalgae cells enhances photosynthesis (Leone, G., De la Cruz Valbuena, G., Cicco, S.R. ''et al.''; Sci Rep 11, 2021; https://www.nature.com/articles/s41598-021-84690-z#Sec10) [[Vključevanje molekularne antene v celice mikroskopsko majhnih kremenastih alg (diatomej) za izboljšanje fotosintetske učinkovitosti]]. Karin Dobravc Škof (20.5.)
# Integrated cascade biorefinery processes for the production of single cell oil by ''Lipomyces starkeyi'' from ''Arundo donax L.'' hydrolysates (Di Fidio N ''et al.''; ''Bioresour. Tecnhol.'', vol.325, p.124635, Apr. 2021.; https://doi.org/10.1016/j.biortech.2020.124635). [[Integrirana procesa biorafinerijske proizvodnje znotrajceličnih založnih lipidov iz Arundo donax L. v Lipomyces starkeyi]] . Katja Doberšek (20.5.)

'''Okoljski vidiki biotehnologije in bioremediacija''' 
# HriGFP Novel Flourescent Protein: Expression and Applications (Saeed, S. ''et al.''; Molecular Biotechnology. 2020; https://link.springer.com/article/10.1007/s12033-020-00243-1) [[ HriGFP fluorescentni protein: Izraz in aplikacije]]. Almina Tahirović (26.5.)
# An extracellular lipase from Amycolatopsis mediterannei is a cutinase with plastic degrading activity (T. Yeqi; Computational and Structural Biotechnology Journal 19; 2021, https://doi.org/10.1016/j.csbj.2021.01.019 ) [[ Izvencelična lipaza iz Amycolatopsis Mediterannei je kutinaza z možnostjo razgradnje plastike.]] Eva Keber (27.05.)
# Nina Lukančič (27.5.)

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T17:43:48Z

Alminatahirovic: /* Matherials and methods */

==Introduction==
Enviromental pollution is one of the greatest global issues to this day and it keeps increasing at an alarming level which poses a great danger to human health. The greatest threats are in fact, industrial wastes and pollutants which are constantly released into the enviroment, including the pesticides that consist of organo-phosphorous compounds or either their derivatives [1]. Those pesticided are often used to increase the food production, even though the high amounts of it can have lethal consequences when it comes to human health or food safety. Since the proper remoting of agricultural areas have been problem for decades, because the usual methods like Chromatography Mass Spectroscopy or Liquid/Gas Chromatography are costly, non-portable and require specialized laboratories, scientist have come up with the idea of using cost-effective portable biosensors which could effectively detect the methyl parathion (one of the organo-phosphorous compounds) on spot [2].

==Green fluorescent protein==
The green fluorescent protein (GFP) is a protein that sucessfully exhibits green fluorescence when exposed to light in the blue to ultraviolet range and GFP usually refers to the protein first isolated from the jellyfish ''Aequorea victoria''. Also, GFP-like coral proteins exhibit a broad spectral diversity ranging from blue to red FPs, so both the stability and expression of it could be optimized by amino acids substitutions possessing different kinds of biophysical properties, therefore such modifications could ensure better rate of maturation and stability of mentioned FPs. Such modificated and more stable monomeric FPs could be used as biosensors [3].

==Matherials and methods==
Materials used in this study were of analytical grade - stock and arsenium solution, both obtained from Sigma. HriGFP gene was cloned into pET28a+vector (containing the T7 lac promoter, induced using isopropyl β-d-1-thiogalactopyranoside) and sucessfully transformed into ''E. coli'' BL21 (DE3) cells. In order to sucessfully transform it in ''Bacillus megaterium'', the gene had to be sub-cloned into the shuttle vector pHIS1522. Kanamycin was added as well, with the role of the resistant marker and the plates were incubated at 37 °C overnight. Structure prediction of the HriGFP was done by homology modeling and entire statistical analysis was performed with Graph-pad Prism. Lastly, all the biosensing experiments were done in total of three biological replicates [1].

==Results==
Research team sucessfully transformed and expressed the HriGFP in ''E. coli'' and ''Bacillus megaterium'' which was presented by fluorescent microscopy. The entire HriGFP structure was modeled with the help of MODELLER - it contained 7 beta sheets, resembling to beta barrel which is specific for GFP molecules. Research team also analyzed the HriGFP interaction with the metal ions where strong interaction was observed with divalent iones, such as Mg and some slightly less strong interactions with iones like Zn and Mn. It was also observed that exposure of HriGFP to the monovalent iones did not show any change in fuorescence intensity and at final, it was concluded that strongest fuorescence quenching was observed in the case of copper as opposed to mercury and arsenic. Alltoghether, the biosensing ability of the HriGFP producing cells was tested and signifcant increase in the fuorescence intensity was observed with the
corresponding decrease in the concentration of methyl parathion, which is considered to be detected from 500 mg to 0.05 mg/L (1.89 mM- 189 μM) concentration [1].

==Conclusion==
HriGFP could be used efciently as biosensor for the detection of heavy metals and harmful organo-phosphorous compounds like methyl-parahtion.

==Literature==

[1] Saeed, S., Mehreen, H., Gerlevik, U., Tariq, A., Manzoor, S., Noreen, Z., Sezerman, U. HriGFP Novel Flourescent Protein: Expression and Applications. ''Molecular Biotechnology''. '''2020''';62: 280-288.

[2] Gong, J., Wang, L., Zhang, L. Electrochemical biosensing of methyl parathion pesticide based on acetylcholinesterase
immobilized onto Au–polypyrrole interlaced network-like nano-composite. ''Biosensors and Bioelectronics''. '''2009''';24: 2285–2288.

[3] Mérola, F., Erard, M., Fredj, A., Pasquier, H. Engineering fuorescent proteins towards ultimate performances: lessons
from the newly developed cyan variants.'' Methods and Applications in Fluorescence''. '''2016''';4: 1200.

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T17:32:44Z

Alminatahirovic: /* Matherials and methods */

==Introduction==
Enviromental pollution is one of the greatest global issues to this day and it keeps increasing at an alarming level which poses a great danger to human health. The greatest threats are in fact, industrial wastes and pollutants which are constantly released into the enviroment, including the pesticides that consist of organo-phosphorous compounds or either their derivatives [1]. Those pesticided are often used to increase the food production, even though the high amounts of it can have lethal consequences when it comes to human health or food safety. Since the proper remoting of agricultural areas have been problem for decades, because the usual methods like Chromatography Mass Spectroscopy or Liquid/Gas Chromatography are costly, non-portable and require specialized laboratories, scientist have come up with the idea of using cost-effective portable biosensors which could effectively detect the methyl parathion (one of the organo-phosphorous compounds) on spot [2].

==Green fluorescent protein==
The green fluorescent protein (GFP) is a protein that sucessfully exhibits green fluorescence when exposed to light in the blue to ultraviolet range and GFP usually refers to the protein first isolated from the jellyfish ''Aequorea victoria''. Also, GFP-like coral proteins exhibit a broad spectral diversity ranging from blue to red FPs, so both the stability and expression of it could be optimized by amino acids substitutions possessing different kinds of biophysical properties, therefore such modifications could ensure better rate of maturation and stability of mentioned FPs. Such modificated and more stable monomeric FPs could be used as biosensors [3].

==Matherials and methods==
Materials used in this study were of analytical grade - stock and arsenium solution, both obtained from Sigma. HriGFP gene was cloned into pET28a+vector (containing the T7 lac promoter, induced using isopropyl β-d-1-thiogalactopyranoside) and sucessfully transformed into ''E. coli'' BL21 (DE3) cells. In order to sucessfully transform it in ''Bacillus megaterium'', the gene had to be sub-cloned into the shuttle vector pHIS1522. Kanamycin was added as well, with the role of the resistant marker and the plates were incubated at 37 °C overnight. Structure prediction of the HriGFP was done by homology modeling and entire statistical analysis was performed with Graph-pad Prism. Lastly, all the biosensing experiments were done in total of three biological replicates [1].

==Literature==

[1] Saeed, S., Mehreen, H., Gerlevik, U., Tariq, A., Manzoor, S., Noreen, Z., Sezerman, U. HriGFP Novel Flourescent Protein: Expression and Applications. ''Molecular Biotechnology''. '''2020''';62: 280-288.

[2] Gong, J., Wang, L., Zhang, L. Electrochemical biosensing of methyl parathion pesticide based on acetylcholinesterase
immobilized onto Au–polypyrrole interlaced network-like nano-composite. ''Biosensors and Bioelectronics''. '''2009''';24: 2285–2288.

[3] Mérola, F., Erard, M., Fredj, A., Pasquier, H. Engineering fuorescent proteins towards ultimate performances: lessons
from the newly developed cyan variants.'' Methods and Applications in Fluorescence''. '''2016''';4: 1200.

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T17:32:35Z

Alminatahirovic: /* Matherials and methods */

==Introduction==
Enviromental pollution is one of the greatest global issues to this day and it keeps increasing at an alarming level which poses a great danger to human health. The greatest threats are in fact, industrial wastes and pollutants which are constantly released into the enviroment, including the pesticides that consist of organo-phosphorous compounds or either their derivatives [1]. Those pesticided are often used to increase the food production, even though the high amounts of it can have lethal consequences when it comes to human health or food safety. Since the proper remoting of agricultural areas have been problem for decades, because the usual methods like Chromatography Mass Spectroscopy or Liquid/Gas Chromatography are costly, non-portable and require specialized laboratories, scientist have come up with the idea of using cost-effective portable biosensors which could effectively detect the methyl parathion (one of the organo-phosphorous compounds) on spot [2].

==Green fluorescent protein==
The green fluorescent protein (GFP) is a protein that sucessfully exhibits green fluorescence when exposed to light in the blue to ultraviolet range and GFP usually refers to the protein first isolated from the jellyfish ''Aequorea victoria''. Also, GFP-like coral proteins exhibit a broad spectral diversity ranging from blue to red FPs, so both the stability and expression of it could be optimized by amino acids substitutions possessing different kinds of biophysical properties, therefore such modifications could ensure better rate of maturation and stability of mentioned FPs. Such modificated and more stable monomeric FPs could be used as biosensors [3].

==Matherials and methods==
Materials used in this study were of analytical grade - stock and arsenium solution, both obtained from Sigma. HriGFP gene was cloned into pET28a+vector (containing the T7 lac promoter, induced using isopropyl β-d-1-thiogalactopyranoside) and sucessfully transformed into ''E. coli'' BL21 (DE3) cells. In order to sucessfully transform it in ''Bacillus megaterium'', the gene had to be sub-cloned into the shuttle vector pHIS1522. Kanamycin was added as well, with the role of the resistant marker and the plates were incubated at 37 °C overnight. Structure prediction of the HriGFP was done by homology modeling and entire statistical analysis was performed with Graph-pad Prism. Lastly, all the biosensing experiments were done in total of three biological replicates [3].

==Literature==

[1] Saeed, S., Mehreen, H., Gerlevik, U., Tariq, A., Manzoor, S., Noreen, Z., Sezerman, U. HriGFP Novel Flourescent Protein: Expression and Applications. ''Molecular Biotechnology''. '''2020''';62: 280-288.

[2] Gong, J., Wang, L., Zhang, L. Electrochemical biosensing of methyl parathion pesticide based on acetylcholinesterase
immobilized onto Au–polypyrrole interlaced network-like nano-composite. ''Biosensors and Bioelectronics''. '''2009''';24: 2285–2288.

[3] Mérola, F., Erard, M., Fredj, A., Pasquier, H. Engineering fuorescent proteins towards ultimate performances: lessons
from the newly developed cyan variants.'' Methods and Applications in Fluorescence''. '''2016''';4: 1200.

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T17:29:59Z

Alminatahirovic: /* Matherials and methods */

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T17:27:55Z

Alminatahirovic: /* Green fluorescent protein */

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T17:24:28Z

Alminatahirovic: /* Literature */

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T17:22:27Z

Alminatahirovic: /* Green fluorescent protein */

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T17:21:56Z

Alminatahirovic: /* Green fluorescent protein */

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T17:21:44Z

Alminatahirovic: /* Introduction */

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T15:02:55Z

Alminatahirovic: /* Literature */

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T15:02:37Z

Alminatahirovic: /* Literature */

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T15:02:30Z

Alminatahirovic: /* Literature */

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T15:00:21Z

Alminatahirovic: /* Literature */

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T15:00:02Z

Alminatahirovic: /* Introduction */

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T14:48:53Z

Alminatahirovic: /* Introduction */

HriGFP fluorescentni protein: Izraz in aplikacije

2021-05-24T14:38:39Z

Alminatahirovic: New page: ==Introduction== Enviromental pollution is one of the greatest global issues to this day and it keeps increasing at an alarming level which poses a great danger to human health. The greate...

Seminarji SB 2020/21

2021-04-19T16:06:43Z

Alminatahirovic:

V študijskem letu 2020/21 študentje 1. letnika predstavljajo naslednje teme:

RAZISKOVALNI ČLANKI

(Vpišite naslov seminarja v slovenščini in ga povežite z novo stranjo, kjer bo povzetek. Na tej novi strani naj bo pod naslovom povezava do izhodiščnega članka na spletu.) 

1 [http://wiki.fkkt.uni-lj.si/index.php/Proizvodnja_1,3-propandiola_iz_različnih_ogljikovodikov_po_nenaravni_poti_preko_3-hidroksipropanojske_kisline Proizvodnja 1,3-propandiola iz različnih ogljikovodikov po nenaravni poti preko 3-hidroksipropanojske kisline]
(Liza Ulčakar) 
2 [http://wiki.fkkt.uni-lj.si/index.php/Doseganje_asimetri%C4%8Dnosti_in_asimetri%C4%8Dne_delitve_pri_E._coli Doseganje asimetričnosti in asimetrične delitve pri E. coli] (Aljaž Bratina) 
3 [http://wiki.fkkt.uni-lj.si/index.php/Zmanj%C5%A1ana_procesivnost_ribosomov_v_sistemu_PURE Zmanjšana procesivnost ribosomov v sistemu PURE] (Tina Kolenc Milavec) 
4 [http://wiki.fkkt.uni-lj.si/index.php/Na%C4%8Drtovana_pot_zvijanja_proteinskih_origamijev Načrtovana pot zvijanja proteinskih origamijev] (Anamarija Agnič) 
5 [http://wiki.fkkt.uni-lj.si/index.php/U%C4%8Dinkovita_svetlobno_inducibilna_Dre_rekombinaza_za_%C4%8Dasovno_in_prostorsko_celi%C4%8Dno_specifi%C4%8Dno_urejanje_genoma_v_mi%C5%A1jih_modelih#VIRI Učinkovita svetlobno inducibilna Dre rekombinaza za časovno in prostorsko celično specifično urejanje genoma v mišjih modelih] (Nika Mikulič Vernik) 
6 [http://wiki.fkkt.uni-lj.si/index.php/Vzpostavitev_termometra_tRNA_za_dolo%C4%8Danje_temperature_optimalne_rasti_mikroorganizmov Vzpostavitev termometra tRNA za določanje temperature optimalne rasti mikroorganizmov] (Urša Štrancar) 
7 [http://wiki.fkkt.uni-lj.si/index.php/Racionalna_zasnova_minimalnih_sinteti%C4%8Dnih_promotorjev_za_rastline#Construction_of_plasmids Racionalna zasnova minimalnih sintetičnih promotorjev za rastline] (Almina Tahirović) 

NAGRAJENI ŠTUDENTSKI PROJEKTI

(Vpišite naslov seminarja v slovenščini in ga povežite z novo stranjo, kjer bo povzetek. Na tej novi strani naj bo pod naslovom povezava do wiki strani študentske ekipe, katere projekt opisujete.) 

1 [http://wiki.fkkt.uni-lj.si/index.php/RESHAPE_-_spreminjanje_morfologije_nitastih_gliv RESHAPE - spreminjanje morfologije nitastih gliv]
(Špela Supej) 
2 [http://wiki.fkkt.uni-lj.si/index.php/The_Chlamy_Cleaner:_razgradnja_pesticida_z_zeleno_algo The Chlamy Cleaner: razgradnja pesticida z zeleno algo] (Doroteja Armič) 
3 [http://wiki.fkkt.uni-lj.si/index.php/TheraPUFA:_nazalni_probiotik_proti_okužbam_in_vnetjem TheraPUFA- nazalni probiotik proti okužbam in vnetjem] (Barbara Slapnik) 
4 [[S-POP: Modularni biosenzor za zaznavanje obstojnih organskih onesnaževal v okoljskih vodah]] (Tadej Medved) 
5 [http://wiki.fkkt.uni-lj.si/index.php/MARS-magnetni_sistem_za_recikliranje_ATP MARS-magnetni sistem za recikliranje ATP] (David Miškić) 
6 [[B.O.T.: Bakterijska oscilacijska terapija za zdravljenje kolorektalnega raka]] (Neža Pavko) 

----

Povzetki v slovenščini naj imajo 1200-1500 besed (viri v to vsoto ne štejejo). Povzetek je treba objaviti dva dni pred predstavitvijo do polnoči (za seminarje v sredo torej v ponedeljek). Predstavitev seminarja naj bo dolga 15 minut (13-17). Sledila bo razprava, ki praviloma ne bo daljša od 5 minut.

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T16:01:28Z

Alminatahirovic: /* Construction of plasmids */

Povzeto po članku: [https://academic.oup.com/nar/article/48/21/11845/5897334 Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48, str. 11845–11856.]

=='''Introduction'''==
Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses. Synthetic transcription factors can be designed by combination of tailored DNA binding domains with effector (activation/repression) domains and nuclear-localization signals. The most widely used activation domain in plants is the acidic VP16 from herpes simplex virus [8].

=='''Identification of candidate transcription factor binding sites (TFBSs)'''==
All expression data for transcription factors encoding genes was obtained from the Expression Atlas [2].

=='''Construction of plasmids'''==
Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site (the cleavage sites of the enzymes which are used in TYPE IIS DNA assembly protocol are usually downstream of the recognition site and can be
composed of any sequence). After ligation, no scar will exist between adjacent fragments. [4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS
terminator [3].

=='''Growth of plant material, protoplast preparation and transfection'''==
The following plant species - ''Arabidopsis thaliana, Brassica rapa, Nicotiana benthamiana'' and ''Hordeum vulgare'' were both germinated and grown in the potting medium under the specific and well controlled circumstances. Photoperiod lasted for 16 horus under the temperature of 22◦C and recorder light intesity was aproximately 160 mol/m2/s [3]. When it comes to the protoplast preparation, they were diluted to 105 ml for process of transfection. Cai et al. followed the protocol written by Yoo et al. which included mixing purified plasmid DNA (4.5 g) and calibrating plasmid together in 96 deep-well (2.2 ml) which contains protoplasts (0.2 ml) [4]. Later, PEG (polyethylene glycol) solution was added to the well, to increase the reacton rate and the overall yield, by mebing a macromolecular crowding agent. They sucessfully produced the transgenic Arabidopsis lines by Agrobacterium mediated tissue transformations and all assembled plasmids were sucessfully transformed into Agrobacterium tumefaciens (they collected the cells by centrifugations and resuspended them into 5% sucrose and seeds from mature siliques). In order to achieve the transgenic lines, all seeds were sterilized with the 70% ethanol for 8-10 minutes and grown on Murashige and Skoog medium [3].

=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==

Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al [7]. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression and images were taken by using a Leica M205FA stereo microsope, whereas the YFP expression was visualized using a SP5 confocal microscope and at last, final images were achieved by using Fiji ImageJ [3].

=='''Results'''==

It is known that genome engineering technology can provide the functions of cis-regulatory elements (CREs) to be dissected and therefore this research tried to describe the analysis, identification and characterization of plant cis-regulatory elements, by revealing their complexity and role in plants regulatory functions. Also, data that is used, has been helpful with predicting the performance of computationally designed minimal synthetic promoters. Therefore, in order to identifiy candidate cis-regulatory elements for use in minimal synthetic constitutive promoters, promoters which have been used for exogenus expressions had been analyzed with promoters from vascular plants included. Promoters from plant-infecting patogens had been included too, such as CaMV35S, Mirabilis Mosaic Virus (MMV) and A. tumefaciens nopaline synthase - AtuNOS. Achieved results had shown that constitutive promoters do indeed have the ability to bind multiple classes of transcription factors. De novo motif identification was performed by using MEME and the presence of a CRE common (C-CRE) was identified, to all pathogen promoters that were included into the studies. Cho et al. also questioned if the positions of the C-CRE's within the promoter is important so the element was relocated to the transcriptional start site and it was reported that the expression was reduced when the element was moved further
in CAMV35S and MMV but no significant impact was reported in the case with AtuNOS, because it is already located distantly to the transcriptional start site. For identification of a functional design for minimal synthetic plant promoters, they had to build and analyze synthetic promoters with a range of level expressions that corespond to the orthogonal transcriptional factors and the primarily design was based on synthetic TALE- responsive elements which consist of 19 bps of random sequence and a second region of variable lenght and a TATA box sequence and a 43 bp minimal core, transcriptional start site included. It was reported that this type of design can be easily used to build promoters with a wide level expression, just by adding different number of binding sites for initial syntetic TALE responsive element. To conclude, promoters from different type of plant pathogens contain a C-CRE which has a significant effect on the expression levels of both natural and synthetic promoters. The utility of minimal synthetic constitutive promoters in genetic circuits was also demonstrated and proven, when Cho et al. constructed a simple multigene constructs where all promoter elements were strictly synthetic and at first, a minimal synthetic constitutive promoters were used just to initiate transcriptional flow by controlling the expression of a otrhogonal transcriptional factos which sucesfully activated the expression of a reporter and indeed, very similiar expression was detected to circuits where the transcriptional factor was controlled by CAMV35S (ised to initiate the process of transcription in transgenic plants). At last, they provided two possible solutions - minimal synthetic constitutive promoters of different strenghts regulated by endogenous transcriptional factos or minimal synthetic constitutive promoter regulated by snythetic orthogonal transcriptional factor. It is yet to be investigated and revealed how the properties of the minimal synthetic constitutive promoters can be modulated when they are combined with different sequence elements [1,3].

=='''Resources'''==
# W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
# R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research. 2020, 44 str. 746 - 752.
# Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48 str. 11845–11856.
# N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.
# S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.
# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.
# G.C. Allen, M.A. Flores-Vergara., S. Krasynanski., S. Kumar et W.F. Thompson: A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 2006, 1 str. 2320–2325.
# S.J. Triezenberg. K.L. LaMarco., S.L. McKnight: Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Deviation. 1988, 2 str. 718-729.

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T16:01:09Z

Alminatahirovic: /* Introduction */

Povzeto po članku: [https://academic.oup.com/nar/article/48/21/11845/5897334 Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48, str. 11845–11856.]

=='''Introduction'''==
Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses. Synthetic transcription factors can be designed by combination of tailored DNA binding domains with effector (activation/repression) domains and nuclear-localization signals. The most widely used activation domain in plants is the acidic VP16 from herpes simplex virus [8].

=='''Identification of candidate transcription factor binding sites (TFBSs)'''==
All expression data for transcription factors encoding genes was obtained from the Expression Atlas [2].

=='''Construction of plasmids'''==
Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site (the cleavage sites of the enzymes which are used in TYPE IIS DNA assembly protocol are usually downstream of the recognition site and can be
composed of any sequence). After ligation, no scar will exist between adjacent assem�bly fragments. [4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS
terminator [3].

=='''Growth of plant material, protoplast preparation and transfection'''==
The following plant species - ''Arabidopsis thaliana, Brassica rapa, Nicotiana benthamiana'' and ''Hordeum vulgare'' were both germinated and grown in the potting medium under the specific and well controlled circumstances. Photoperiod lasted for 16 horus under the temperature of 22◦C and recorder light intesity was aproximately 160 mol/m2/s [3]. When it comes to the protoplast preparation, they were diluted to 105 ml for process of transfection. Cai et al. followed the protocol written by Yoo et al. which included mixing purified plasmid DNA (4.5 g) and calibrating plasmid together in 96 deep-well (2.2 ml) which contains protoplasts (0.2 ml) [4]. Later, PEG (polyethylene glycol) solution was added to the well, to increase the reacton rate and the overall yield, by mebing a macromolecular crowding agent. They sucessfully produced the transgenic Arabidopsis lines by Agrobacterium mediated tissue transformations and all assembled plasmids were sucessfully transformed into Agrobacterium tumefaciens (they collected the cells by centrifugations and resuspended them into 5% sucrose and seeds from mature siliques). In order to achieve the transgenic lines, all seeds were sterilized with the 70% ethanol for 8-10 minutes and grown on Murashige and Skoog medium [3].

=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==

Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al [7]. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression and images were taken by using a Leica M205FA stereo microsope, whereas the YFP expression was visualized using a SP5 confocal microscope and at last, final images were achieved by using Fiji ImageJ [3].

=='''Results'''==

It is known that genome engineering technology can provide the functions of cis-regulatory elements (CREs) to be dissected and therefore this research tried to describe the analysis, identification and characterization of plant cis-regulatory elements, by revealing their complexity and role in plants regulatory functions. Also, data that is used, has been helpful with predicting the performance of computationally designed minimal synthetic promoters. Therefore, in order to identifiy candidate cis-regulatory elements for use in minimal synthetic constitutive promoters, promoters which have been used for exogenus expressions had been analyzed with promoters from vascular plants included. Promoters from plant-infecting patogens had been included too, such as CaMV35S, Mirabilis Mosaic Virus (MMV) and A. tumefaciens nopaline synthase - AtuNOS. Achieved results had shown that constitutive promoters do indeed have the ability to bind multiple classes of transcription factors. De novo motif identification was performed by using MEME and the presence of a CRE common (C-CRE) was identified, to all pathogen promoters that were included into the studies. Cho et al. also questioned if the positions of the C-CRE's within the promoter is important so the element was relocated to the transcriptional start site and it was reported that the expression was reduced when the element was moved further
in CAMV35S and MMV but no significant impact was reported in the case with AtuNOS, because it is already located distantly to the transcriptional start site. For identification of a functional design for minimal synthetic plant promoters, they had to build and analyze synthetic promoters with a range of level expressions that corespond to the orthogonal transcriptional factors and the primarily design was based on synthetic TALE- responsive elements which consist of 19 bps of random sequence and a second region of variable lenght and a TATA box sequence and a 43 bp minimal core, transcriptional start site included. It was reported that this type of design can be easily used to build promoters with a wide level expression, just by adding different number of binding sites for initial syntetic TALE responsive element. To conclude, promoters from different type of plant pathogens contain a C-CRE which has a significant effect on the expression levels of both natural and synthetic promoters. The utility of minimal synthetic constitutive promoters in genetic circuits was also demonstrated and proven, when Cho et al. constructed a simple multigene constructs where all promoter elements were strictly synthetic and at first, a minimal synthetic constitutive promoters were used just to initiate transcriptional flow by controlling the expression of a otrhogonal transcriptional factos which sucesfully activated the expression of a reporter and indeed, very similiar expression was detected to circuits where the transcriptional factor was controlled by CAMV35S (ised to initiate the process of transcription in transgenic plants). At last, they provided two possible solutions - minimal synthetic constitutive promoters of different strenghts regulated by endogenous transcriptional factos or minimal synthetic constitutive promoter regulated by snythetic orthogonal transcriptional factor. It is yet to be investigated and revealed how the properties of the minimal synthetic constitutive promoters can be modulated when they are combined with different sequence elements [1,3].

=='''Resources'''==
# W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
# R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research. 2020, 44 str. 746 - 752.
# Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48 str. 11845–11856.
# N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.
# S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.
# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.
# G.C. Allen, M.A. Flores-Vergara., S. Krasynanski., S. Kumar et W.F. Thompson: A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 2006, 1 str. 2320–2325.
# S.J. Triezenberg. K.L. LaMarco., S.L. McKnight: Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Deviation. 1988, 2 str. 718-729.

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T16:00:03Z

Alminatahirovic: /* Resources */

Povzeto po članku: [https://academic.oup.com/nar/article/48/21/11845/5897334 Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48, str. 11845–11856.]

=='''Introduction'''==
Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses. Synthetic transcription factors can be designed by combination of tailored DNA binding domains with effector (activation/repression) domains and nuclear-localization signals. The most widely used activation domain in plants is the acidic VP16 from herpes simplex virus [8

=='''Identification of candidate transcription factor binding sites (TFBSs)'''==
All expression data for transcription factors encoding genes was obtained from the Expression Atlas [2].

=='''Construction of plasmids'''==
Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site (the cleavage sites of the enzymes which are used in TYPE IIS DNA assembly protocol are usually downstream of the recognition site and can be
composed of any sequence). After ligation, no scar will exist between adjacent assem�bly fragments. [4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS
terminator [3].

=='''Growth of plant material, protoplast preparation and transfection'''==
The following plant species - ''Arabidopsis thaliana, Brassica rapa, Nicotiana benthamiana'' and ''Hordeum vulgare'' were both germinated and grown in the potting medium under the specific and well controlled circumstances. Photoperiod lasted for 16 horus under the temperature of 22◦C and recorder light intesity was aproximately 160 mol/m2/s [3]. When it comes to the protoplast preparation, they were diluted to 105 ml for process of transfection. Cai et al. followed the protocol written by Yoo et al. which included mixing purified plasmid DNA (4.5 g) and calibrating plasmid together in 96 deep-well (2.2 ml) which contains protoplasts (0.2 ml) [4]. Later, PEG (polyethylene glycol) solution was added to the well, to increase the reacton rate and the overall yield, by mebing a macromolecular crowding agent. They sucessfully produced the transgenic Arabidopsis lines by Agrobacterium mediated tissue transformations and all assembled plasmids were sucessfully transformed into Agrobacterium tumefaciens (they collected the cells by centrifugations and resuspended them into 5% sucrose and seeds from mature siliques). In order to achieve the transgenic lines, all seeds were sterilized with the 70% ethanol for 8-10 minutes and grown on Murashige and Skoog medium [3].

=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==

Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al [7]. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression and images were taken by using a Leica M205FA stereo microsope, whereas the YFP expression was visualized using a SP5 confocal microscope and at last, final images were achieved by using Fiji ImageJ [3].

=='''Results'''==

It is known that genome engineering technology can provide the functions of cis-regulatory elements (CREs) to be dissected and therefore this research tried to describe the analysis, identification and characterization of plant cis-regulatory elements, by revealing their complexity and role in plants regulatory functions. Also, data that is used, has been helpful with predicting the performance of computationally designed minimal synthetic promoters. Therefore, in order to identifiy candidate cis-regulatory elements for use in minimal synthetic constitutive promoters, promoters which have been used for exogenus expressions had been analyzed with promoters from vascular plants included. Promoters from plant-infecting patogens had been included too, such as CaMV35S, Mirabilis Mosaic Virus (MMV) and A. tumefaciens nopaline synthase - AtuNOS. Achieved results had shown that constitutive promoters do indeed have the ability to bind multiple classes of transcription factors. De novo motif identification was performed by using MEME and the presence of a CRE common (C-CRE) was identified, to all pathogen promoters that were included into the studies. Cho et al. also questioned if the positions of the C-CRE's within the promoter is important so the element was relocated to the transcriptional start site and it was reported that the expression was reduced when the element was moved further
in CAMV35S and MMV but no significant impact was reported in the case with AtuNOS, because it is already located distantly to the transcriptional start site. For identification of a functional design for minimal synthetic plant promoters, they had to build and analyze synthetic promoters with a range of level expressions that corespond to the orthogonal transcriptional factors and the primarily design was based on synthetic TALE- responsive elements which consist of 19 bps of random sequence and a second region of variable lenght and a TATA box sequence and a 43 bp minimal core, transcriptional start site included. It was reported that this type of design can be easily used to build promoters with a wide level expression, just by adding different number of binding sites for initial syntetic TALE responsive element. To conclude, promoters from different type of plant pathogens contain a C-CRE which has a significant effect on the expression levels of both natural and synthetic promoters. The utility of minimal synthetic constitutive promoters in genetic circuits was also demonstrated and proven, when Cho et al. constructed a simple multigene constructs where all promoter elements were strictly synthetic and at first, a minimal synthetic constitutive promoters were used just to initiate transcriptional flow by controlling the expression of a otrhogonal transcriptional factos which sucesfully activated the expression of a reporter and indeed, very similiar expression was detected to circuits where the transcriptional factor was controlled by CAMV35S (ised to initiate the process of transcription in transgenic plants). At last, they provided two possible solutions - minimal synthetic constitutive promoters of different strenghts regulated by endogenous transcriptional factos or minimal synthetic constitutive promoter regulated by snythetic orthogonal transcriptional factor. It is yet to be investigated and revealed how the properties of the minimal synthetic constitutive promoters can be modulated when they are combined with different sequence elements [1,3].

=='''Resources'''==
# W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
# R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research. 2020, 44 str. 746 - 752.
# Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48 str. 11845–11856.
# N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.
# S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.
# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.
# G.C. Allen, M.A. Flores-Vergara., S. Krasynanski., S. Kumar et W.F. Thompson: A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 2006, 1 str. 2320–2325.
# S.J. Triezenberg. K.L. LaMarco., S.L. McKnight: Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Deviation. 1988, 2 str. 718-729.

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T15:59:38Z

Alminatahirovic: /* Resources */

Povzeto po članku: [https://academic.oup.com/nar/article/48/21/11845/5897334 Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48, str. 11845–11856.]

=='''Introduction'''==
Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses. Synthetic transcription factors can be designed by combination of tailored DNA binding domains with effector (activation/repression) domains and nuclear-localization signals. The most widely used activation domain in plants is the acidic VP16 from herpes simplex virus [8

=='''Identification of candidate transcription factor binding sites (TFBSs)'''==
All expression data for transcription factors encoding genes was obtained from the Expression Atlas [2].

=='''Construction of plasmids'''==
Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site (the cleavage sites of the enzymes which are used in TYPE IIS DNA assembly protocol are usually downstream of the recognition site and can be
composed of any sequence). After ligation, no scar will exist between adjacent assem�bly fragments. [4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS
terminator [3].

=='''Growth of plant material, protoplast preparation and transfection'''==
The following plant species - ''Arabidopsis thaliana, Brassica rapa, Nicotiana benthamiana'' and ''Hordeum vulgare'' were both germinated and grown in the potting medium under the specific and well controlled circumstances. Photoperiod lasted for 16 horus under the temperature of 22◦C and recorder light intesity was aproximately 160 mol/m2/s [3]. When it comes to the protoplast preparation, they were diluted to 105 ml for process of transfection. Cai et al. followed the protocol written by Yoo et al. which included mixing purified plasmid DNA (4.5 g) and calibrating plasmid together in 96 deep-well (2.2 ml) which contains protoplasts (0.2 ml) [4]. Later, PEG (polyethylene glycol) solution was added to the well, to increase the reacton rate and the overall yield, by mebing a macromolecular crowding agent. They sucessfully produced the transgenic Arabidopsis lines by Agrobacterium mediated tissue transformations and all assembled plasmids were sucessfully transformed into Agrobacterium tumefaciens (they collected the cells by centrifugations and resuspended them into 5% sucrose and seeds from mature siliques). In order to achieve the transgenic lines, all seeds were sterilized with the 70% ethanol for 8-10 minutes and grown on Murashige and Skoog medium [3].

=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==

Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al [7]. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression and images were taken by using a Leica M205FA stereo microsope, whereas the YFP expression was visualized using a SP5 confocal microscope and at last, final images were achieved by using Fiji ImageJ [3].

=='''Results'''==

It is known that genome engineering technology can provide the functions of cis-regulatory elements (CREs) to be dissected and therefore this research tried to describe the analysis, identification and characterization of plant cis-regulatory elements, by revealing their complexity and role in plants regulatory functions. Also, data that is used, has been helpful with predicting the performance of computationally designed minimal synthetic promoters. Therefore, in order to identifiy candidate cis-regulatory elements for use in minimal synthetic constitutive promoters, promoters which have been used for exogenus expressions had been analyzed with promoters from vascular plants included. Promoters from plant-infecting patogens had been included too, such as CaMV35S, Mirabilis Mosaic Virus (MMV) and A. tumefaciens nopaline synthase - AtuNOS. Achieved results had shown that constitutive promoters do indeed have the ability to bind multiple classes of transcription factors. De novo motif identification was performed by using MEME and the presence of a CRE common (C-CRE) was identified, to all pathogen promoters that were included into the studies. Cho et al. also questioned if the positions of the C-CRE's within the promoter is important so the element was relocated to the transcriptional start site and it was reported that the expression was reduced when the element was moved further
in CAMV35S and MMV but no significant impact was reported in the case with AtuNOS, because it is already located distantly to the transcriptional start site. For identification of a functional design for minimal synthetic plant promoters, they had to build and analyze synthetic promoters with a range of level expressions that corespond to the orthogonal transcriptional factors and the primarily design was based on synthetic TALE- responsive elements which consist of 19 bps of random sequence and a second region of variable lenght and a TATA box sequence and a 43 bp minimal core, transcriptional start site included. It was reported that this type of design can be easily used to build promoters with a wide level expression, just by adding different number of binding sites for initial syntetic TALE responsive element. To conclude, promoters from different type of plant pathogens contain a C-CRE which has a significant effect on the expression levels of both natural and synthetic promoters. The utility of minimal synthetic constitutive promoters in genetic circuits was also demonstrated and proven, when Cho et al. constructed a simple multigene constructs where all promoter elements were strictly synthetic and at first, a minimal synthetic constitutive promoters were used just to initiate transcriptional flow by controlling the expression of a otrhogonal transcriptional factos which sucesfully activated the expression of a reporter and indeed, very similiar expression was detected to circuits where the transcriptional factor was controlled by CAMV35S (ised to initiate the process of transcription in transgenic plants). At last, they provided two possible solutions - minimal synthetic constitutive promoters of different strenghts regulated by endogenous transcriptional factos or minimal synthetic constitutive promoter regulated by snythetic orthogonal transcriptional factor. It is yet to be investigated and revealed how the properties of the minimal synthetic constitutive promoters can be modulated when they are combined with different sequence elements [1,3].

=='''Resources'''==
# W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
# R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research, 44 str. 746 - 752.
# Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48 str. 11845–11856.
# N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.
# S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.
# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.
# G.C. Allen, M.A. Flores-Vergara., S. Krasynanski., S. Kumar et W.F. Thompson: A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 2006, 1 str. 2320–2325.
# S.J. Triezenberg. K.L. LaMarco., S.L. McKnight: Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Deviation. 1988, 2 str. 718-729.

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T15:59:29Z

Alminatahirovic: /* Resources */

Povzeto po članku: [https://academic.oup.com/nar/article/48/21/11845/5897334 Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48, str. 11845–11856.]

=='''Introduction'''==
Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses. Synthetic transcription factors can be designed by combination of tailored DNA binding domains with effector (activation/repression) domains and nuclear-localization signals. The most widely used activation domain in plants is the acidic VP16 from herpes simplex virus [8

=='''Identification of candidate transcription factor binding sites (TFBSs)'''==
All expression data for transcription factors encoding genes was obtained from the Expression Atlas [2].

=='''Construction of plasmids'''==
Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site (the cleavage sites of the enzymes which are used in TYPE IIS DNA assembly protocol are usually downstream of the recognition site and can be
composed of any sequence). After ligation, no scar will exist between adjacent assem�bly fragments. [4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS
terminator [3].

=='''Growth of plant material, protoplast preparation and transfection'''==
The following plant species - ''Arabidopsis thaliana, Brassica rapa, Nicotiana benthamiana'' and ''Hordeum vulgare'' were both germinated and grown in the potting medium under the specific and well controlled circumstances. Photoperiod lasted for 16 horus under the temperature of 22◦C and recorder light intesity was aproximately 160 mol/m2/s [3]. When it comes to the protoplast preparation, they were diluted to 105 ml for process of transfection. Cai et al. followed the protocol written by Yoo et al. which included mixing purified plasmid DNA (4.5 g) and calibrating plasmid together in 96 deep-well (2.2 ml) which contains protoplasts (0.2 ml) [4]. Later, PEG (polyethylene glycol) solution was added to the well, to increase the reacton rate and the overall yield, by mebing a macromolecular crowding agent. They sucessfully produced the transgenic Arabidopsis lines by Agrobacterium mediated tissue transformations and all assembled plasmids were sucessfully transformed into Agrobacterium tumefaciens (they collected the cells by centrifugations and resuspended them into 5% sucrose and seeds from mature siliques). In order to achieve the transgenic lines, all seeds were sterilized with the 70% ethanol for 8-10 minutes and grown on Murashige and Skoog medium [3].

=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==

Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al [7]. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression and images were taken by using a Leica M205FA stereo microsope, whereas the YFP expression was visualized using a SP5 confocal microscope and at last, final images were achieved by using Fiji ImageJ [3].

=='''Results'''==

It is known that genome engineering technology can provide the functions of cis-regulatory elements (CREs) to be dissected and therefore this research tried to describe the analysis, identification and characterization of plant cis-regulatory elements, by revealing their complexity and role in plants regulatory functions. Also, data that is used, has been helpful with predicting the performance of computationally designed minimal synthetic promoters. Therefore, in order to identifiy candidate cis-regulatory elements for use in minimal synthetic constitutive promoters, promoters which have been used for exogenus expressions had been analyzed with promoters from vascular plants included. Promoters from plant-infecting patogens had been included too, such as CaMV35S, Mirabilis Mosaic Virus (MMV) and A. tumefaciens nopaline synthase - AtuNOS. Achieved results had shown that constitutive promoters do indeed have the ability to bind multiple classes of transcription factors. De novo motif identification was performed by using MEME and the presence of a CRE common (C-CRE) was identified, to all pathogen promoters that were included into the studies. Cho et al. also questioned if the positions of the C-CRE's within the promoter is important so the element was relocated to the transcriptional start site and it was reported that the expression was reduced when the element was moved further
in CAMV35S and MMV but no significant impact was reported in the case with AtuNOS, because it is already located distantly to the transcriptional start site. For identification of a functional design for minimal synthetic plant promoters, they had to build and analyze synthetic promoters with a range of level expressions that corespond to the orthogonal transcriptional factors and the primarily design was based on synthetic TALE- responsive elements which consist of 19 bps of random sequence and a second region of variable lenght and a TATA box sequence and a 43 bp minimal core, transcriptional start site included. It was reported that this type of design can be easily used to build promoters with a wide level expression, just by adding different number of binding sites for initial syntetic TALE responsive element. To conclude, promoters from different type of plant pathogens contain a C-CRE which has a significant effect on the expression levels of both natural and synthetic promoters. The utility of minimal synthetic constitutive promoters in genetic circuits was also demonstrated and proven, when Cho et al. constructed a simple multigene constructs where all promoter elements were strictly synthetic and at first, a minimal synthetic constitutive promoters were used just to initiate transcriptional flow by controlling the expression of a otrhogonal transcriptional factos which sucesfully activated the expression of a reporter and indeed, very similiar expression was detected to circuits where the transcriptional factor was controlled by CAMV35S (ised to initiate the process of transcription in transgenic plants). At last, they provided two possible solutions - minimal synthetic constitutive promoters of different strenghts regulated by endogenous transcriptional factos or minimal synthetic constitutive promoter regulated by snythetic orthogonal transcriptional factor. It is yet to be investigated and revealed how the properties of the minimal synthetic constitutive promoters can be modulated when they are combined with different sequence elements [1,3].

=='''Resources'''==
# W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
# R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research, 44 str. 746 - 752.
# Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48 str. 11845–11856.
# N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.
# S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.
# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.
# G.C. Allen, M.A. Flores-Vergara., S. Krasynanski., S. Kumar et W.F. Thompson: A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 2006, 1 str. 2320–2325.
# S.J. Triezenberg. K.L. LaMarco., S.L. McKnight: Functional dissection of VP16, the trans-activator of herpes simplex virus
immediate early gene expression. Genes Deviation. 1988, 2 str. 718-729.

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T15:58:27Z

Alminatahirovic: /* Introduction */

Povzeto po članku: [https://academic.oup.com/nar/article/48/21/11845/5897334 Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48, str. 11845–11856.]

=='''Introduction'''==
Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses. Synthetic transcription factors can be designed by combination of tailored DNA binding domains with effector (activation/repression) domains and nuclear-localization signals. The most widely used activation domain in plants is the acidic VP16 from herpes simplex virus [8

=='''Identification of candidate transcription factor binding sites (TFBSs)'''==
All expression data for transcription factors encoding genes was obtained from the Expression Atlas [2].

=='''Construction of plasmids'''==
Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site (the cleavage sites of the enzymes which are used in TYPE IIS DNA assembly protocol are usually downstream of the recognition site and can be
composed of any sequence). After ligation, no scar will exist between adjacent assem�bly fragments. [4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS
terminator [3].

=='''Growth of plant material, protoplast preparation and transfection'''==
The following plant species - ''Arabidopsis thaliana, Brassica rapa, Nicotiana benthamiana'' and ''Hordeum vulgare'' were both germinated and grown in the potting medium under the specific and well controlled circumstances. Photoperiod lasted for 16 horus under the temperature of 22◦C and recorder light intesity was aproximately 160 mol/m2/s [3]. When it comes to the protoplast preparation, they were diluted to 105 ml for process of transfection. Cai et al. followed the protocol written by Yoo et al. which included mixing purified plasmid DNA (4.5 g) and calibrating plasmid together in 96 deep-well (2.2 ml) which contains protoplasts (0.2 ml) [4]. Later, PEG (polyethylene glycol) solution was added to the well, to increase the reacton rate and the overall yield, by mebing a macromolecular crowding agent. They sucessfully produced the transgenic Arabidopsis lines by Agrobacterium mediated tissue transformations and all assembled plasmids were sucessfully transformed into Agrobacterium tumefaciens (they collected the cells by centrifugations and resuspended them into 5% sucrose and seeds from mature siliques). In order to achieve the transgenic lines, all seeds were sterilized with the 70% ethanol for 8-10 minutes and grown on Murashige and Skoog medium [3].

=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==

Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al [7]. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression and images were taken by using a Leica M205FA stereo microsope, whereas the YFP expression was visualized using a SP5 confocal microscope and at last, final images were achieved by using Fiji ImageJ [3].

=='''Results'''==

It is known that genome engineering technology can provide the functions of cis-regulatory elements (CREs) to be dissected and therefore this research tried to describe the analysis, identification and characterization of plant cis-regulatory elements, by revealing their complexity and role in plants regulatory functions. Also, data that is used, has been helpful with predicting the performance of computationally designed minimal synthetic promoters. Therefore, in order to identifiy candidate cis-regulatory elements for use in minimal synthetic constitutive promoters, promoters which have been used for exogenus expressions had been analyzed with promoters from vascular plants included. Promoters from plant-infecting patogens had been included too, such as CaMV35S, Mirabilis Mosaic Virus (MMV) and A. tumefaciens nopaline synthase - AtuNOS. Achieved results had shown that constitutive promoters do indeed have the ability to bind multiple classes of transcription factors. De novo motif identification was performed by using MEME and the presence of a CRE common (C-CRE) was identified, to all pathogen promoters that were included into the studies. Cho et al. also questioned if the positions of the C-CRE's within the promoter is important so the element was relocated to the transcriptional start site and it was reported that the expression was reduced when the element was moved further
in CAMV35S and MMV but no significant impact was reported in the case with AtuNOS, because it is already located distantly to the transcriptional start site. For identification of a functional design for minimal synthetic plant promoters, they had to build and analyze synthetic promoters with a range of level expressions that corespond to the orthogonal transcriptional factors and the primarily design was based on synthetic TALE- responsive elements which consist of 19 bps of random sequence and a second region of variable lenght and a TATA box sequence and a 43 bp minimal core, transcriptional start site included. It was reported that this type of design can be easily used to build promoters with a wide level expression, just by adding different number of binding sites for initial syntetic TALE responsive element. To conclude, promoters from different type of plant pathogens contain a C-CRE which has a significant effect on the expression levels of both natural and synthetic promoters. The utility of minimal synthetic constitutive promoters in genetic circuits was also demonstrated and proven, when Cho et al. constructed a simple multigene constructs where all promoter elements were strictly synthetic and at first, a minimal synthetic constitutive promoters were used just to initiate transcriptional flow by controlling the expression of a otrhogonal transcriptional factos which sucesfully activated the expression of a reporter and indeed, very similiar expression was detected to circuits where the transcriptional factor was controlled by CAMV35S (ised to initiate the process of transcription in transgenic plants). At last, they provided two possible solutions - minimal synthetic constitutive promoters of different strenghts regulated by endogenous transcriptional factos or minimal synthetic constitutive promoter regulated by snythetic orthogonal transcriptional factor. It is yet to be investigated and revealed how the properties of the minimal synthetic constitutive promoters can be modulated when they are combined with different sequence elements [1,3].

=='''Resources'''==
# W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
# R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research, 44 str. 746 - 752.
# Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48 str. 11845–11856.
# N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.
# S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.
# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.
# G.C. Allen, M.A. Flores-Vergara., S. Krasynanski., S. Kumar et W.F. Thompson: A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 2006, 1 str. 2320–2325.

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T15:56:30Z

Alminatahirovic: /* Construction of plasmids */

Povzeto po članku: [https://academic.oup.com/nar/article/48/21/11845/5897334 Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48, str. 11845–11856.]

=='''Introduction'''==
Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses [6].

=='''Identification of candidate transcription factor binding sites (TFBSs)'''==
All expression data for transcription factors encoding genes was obtained from the Expression Atlas [2].

=='''Construction of plasmids'''==
Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site (the cleavage sites of the enzymes which are used in TYPE IIS DNA assembly protocol are usually downstream of the recognition site and can be
composed of any sequence). After ligation, no scar will exist between adjacent assem�bly fragments. [4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS
terminator [3].

=='''Growth of plant material, protoplast preparation and transfection'''==
The following plant species - ''Arabidopsis thaliana, Brassica rapa, Nicotiana benthamiana'' and ''Hordeum vulgare'' were both germinated and grown in the potting medium under the specific and well controlled circumstances. Photoperiod lasted for 16 horus under the temperature of 22◦C and recorder light intesity was aproximately 160 mol/m2/s [3]. When it comes to the protoplast preparation, they were diluted to 105 ml for process of transfection. Cai et al. followed the protocol written by Yoo et al. which included mixing purified plasmid DNA (4.5 g) and calibrating plasmid together in 96 deep-well (2.2 ml) which contains protoplasts (0.2 ml) [4]. Later, PEG (polyethylene glycol) solution was added to the well, to increase the reacton rate and the overall yield, by mebing a macromolecular crowding agent. They sucessfully produced the transgenic Arabidopsis lines by Agrobacterium mediated tissue transformations and all assembled plasmids were sucessfully transformed into Agrobacterium tumefaciens (they collected the cells by centrifugations and resuspended them into 5% sucrose and seeds from mature siliques). In order to achieve the transgenic lines, all seeds were sterilized with the 70% ethanol for 8-10 minutes and grown on Murashige and Skoog medium [3].

=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==

Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al [7]. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression and images were taken by using a Leica M205FA stereo microsope, whereas the YFP expression was visualized using a SP5 confocal microscope and at last, final images were achieved by using Fiji ImageJ [3].

=='''Results'''==

It is known that genome engineering technology can provide the functions of cis-regulatory elements (CREs) to be dissected and therefore this research tried to describe the analysis, identification and characterization of plant cis-regulatory elements, by revealing their complexity and role in plants regulatory functions. Also, data that is used, has been helpful with predicting the performance of computationally designed minimal synthetic promoters. Therefore, in order to identifiy candidate cis-regulatory elements for use in minimal synthetic constitutive promoters, promoters which have been used for exogenus expressions had been analyzed with promoters from vascular plants included. Promoters from plant-infecting patogens had been included too, such as CaMV35S, Mirabilis Mosaic Virus (MMV) and A. tumefaciens nopaline synthase - AtuNOS. Achieved results had shown that constitutive promoters do indeed have the ability to bind multiple classes of transcription factors. De novo motif identification was performed by using MEME and the presence of a CRE common (C-CRE) was identified, to all pathogen promoters that were included into the studies. Cho et al. also questioned if the positions of the C-CRE's within the promoter is important so the element was relocated to the transcriptional start site and it was reported that the expression was reduced when the element was moved further
in CAMV35S and MMV but no significant impact was reported in the case with AtuNOS, because it is already located distantly to the transcriptional start site. For identification of a functional design for minimal synthetic plant promoters, they had to build and analyze synthetic promoters with a range of level expressions that corespond to the orthogonal transcriptional factors and the primarily design was based on synthetic TALE- responsive elements which consist of 19 bps of random sequence and a second region of variable lenght and a TATA box sequence and a 43 bp minimal core, transcriptional start site included. It was reported that this type of design can be easily used to build promoters with a wide level expression, just by adding different number of binding sites for initial syntetic TALE responsive element. To conclude, promoters from different type of plant pathogens contain a C-CRE which has a significant effect on the expression levels of both natural and synthetic promoters. The utility of minimal synthetic constitutive promoters in genetic circuits was also demonstrated and proven, when Cho et al. constructed a simple multigene constructs where all promoter elements were strictly synthetic and at first, a minimal synthetic constitutive promoters were used just to initiate transcriptional flow by controlling the expression of a otrhogonal transcriptional factos which sucesfully activated the expression of a reporter and indeed, very similiar expression was detected to circuits where the transcriptional factor was controlled by CAMV35S (ised to initiate the process of transcription in transgenic plants). At last, they provided two possible solutions - minimal synthetic constitutive promoters of different strenghts regulated by endogenous transcriptional factos or minimal synthetic constitutive promoter regulated by snythetic orthogonal transcriptional factor. It is yet to be investigated and revealed how the properties of the minimal synthetic constitutive promoters can be modulated when they are combined with different sequence elements [1,3].

=='''Resources'''==
# W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
# R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research, 44 str. 746 - 752.
# Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48 str. 11845–11856.
# N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.
# S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.
# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.
# G.C. Allen, M.A. Flores-Vergara., S. Krasynanski., S. Kumar et W.F. Thompson: A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 2006, 1 str. 2320–2325.

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T15:51:43Z

Alminatahirovic: /* Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression */

Povzeto po članku: [https://academic.oup.com/nar/article/48/21/11845/5897334 Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48, str. 11845–11856.]

=='''Introduction'''==
Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses [6].

=='''Identification of candidate transcription factor binding sites (TFBSs)'''==
All expression data for transcription factors encoding genes was obtained from the Expression Atlas [2].

=='''Construction of plasmids'''==
Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site)[4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS
terminator [3].

=='''Growth of plant material, protoplast preparation and transfection'''==
The following plant species - ''Arabidopsis thaliana, Brassica rapa, Nicotiana benthamiana'' and ''Hordeum vulgare'' were both germinated and grown in the potting medium under the specific and well controlled circumstances. Photoperiod lasted for 16 horus under the temperature of 22◦C and recorder light intesity was aproximately 160 mol/m2/s [3]. When it comes to the protoplast preparation, they were diluted to 105 ml for process of transfection. Cai et al. followed the protocol written by Yoo et al. which included mixing purified plasmid DNA (4.5 g) and calibrating plasmid together in 96 deep-well (2.2 ml) which contains protoplasts (0.2 ml) [4]. Later, PEG (polyethylene glycol) solution was added to the well, to increase the reacton rate and the overall yield, by mebing a macromolecular crowding agent. They sucessfully produced the transgenic Arabidopsis lines by Agrobacterium mediated tissue transformations and all assembled plasmids were sucessfully transformed into Agrobacterium tumefaciens (they collected the cells by centrifugations and resuspended them into 5% sucrose and seeds from mature siliques). In order to achieve the transgenic lines, all seeds were sterilized with the 70% ethanol for 8-10 minutes and grown on Murashige and Skoog medium [3].

=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==

Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al [7]. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression and images were taken by using a Leica M205FA stereo microsope, whereas the YFP expression was visualized using a SP5 confocal microscope and at last, final images were achieved by using Fiji ImageJ [3].

=='''Results'''==

It is known that genome engineering technology can provide the functions of cis-regulatory elements (CREs) to be dissected and therefore this research tried to describe the analysis, identification and characterization of plant cis-regulatory elements, by revealing their complexity and role in plants regulatory functions. Also, data that is used, has been helpful with predicting the performance of computationally designed minimal synthetic promoters. Therefore, in order to identifiy candidate cis-regulatory elements for use in minimal synthetic constitutive promoters, promoters which have been used for exogenus expressions had been analyzed with promoters from vascular plants included. Promoters from plant-infecting patogens had been included too, such as CaMV35S, Mirabilis Mosaic Virus (MMV) and A. tumefaciens nopaline synthase - AtuNOS. Achieved results had shown that constitutive promoters do indeed have the ability to bind multiple classes of transcription factors. De novo motif identification was performed by using MEME and the presence of a CRE common (C-CRE) was identified, to all pathogen promoters that were included into the studies. Cho et al. also questioned if the positions of the C-CRE's within the promoter is important so the element was relocated to the transcriptional start site and it was reported that the expression was reduced when the element was moved further
in CAMV35S and MMV but no significant impact was reported in the case with AtuNOS, because it is already located distantly to the transcriptional start site. For identification of a functional design for minimal synthetic plant promoters, they had to build and analyze synthetic promoters with a range of level expressions that corespond to the orthogonal transcriptional factors and the primarily design was based on synthetic TALE- responsive elements which consist of 19 bps of random sequence and a second region of variable lenght and a TATA box sequence and a 43 bp minimal core, transcriptional start site included. It was reported that this type of design can be easily used to build promoters with a wide level expression, just by adding different number of binding sites for initial syntetic TALE responsive element. To conclude, promoters from different type of plant pathogens contain a C-CRE which has a significant effect on the expression levels of both natural and synthetic promoters. The utility of minimal synthetic constitutive promoters in genetic circuits was also demonstrated and proven, when Cho et al. constructed a simple multigene constructs where all promoter elements were strictly synthetic and at first, a minimal synthetic constitutive promoters were used just to initiate transcriptional flow by controlling the expression of a otrhogonal transcriptional factos which sucesfully activated the expression of a reporter and indeed, very similiar expression was detected to circuits where the transcriptional factor was controlled by CAMV35S (ised to initiate the process of transcription in transgenic plants). At last, they provided two possible solutions - minimal synthetic constitutive promoters of different strenghts regulated by endogenous transcriptional factos or minimal synthetic constitutive promoter regulated by snythetic orthogonal transcriptional factor. It is yet to be investigated and revealed how the properties of the minimal synthetic constitutive promoters can be modulated when they are combined with different sequence elements [1,3].

=='''Resources'''==
# W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
# R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research, 44 str. 746 - 752.
# Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48 str. 11845–11856.
# N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.
# S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.
# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.
# G.C. Allen, M.A. Flores-Vergara., S. Krasynanski., S. Kumar et W.F. Thompson: A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 2006, 1 str. 2320–2325.

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T15:47:48Z

Alminatahirovic: /* Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression */

Povzeto po članku: [https://academic.oup.com/nar/article/48/21/11845/5897334 Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48, str. 11845–11856.]

=='''Introduction'''==
Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses [6].

=='''Identification of candidate transcription factor binding sites (TFBSs)'''==
All expression data for transcription factors encoding genes was obtained from the Expression Atlas [2].

=='''Construction of plasmids'''==
Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site)[4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS
terminator [3].

=='''Growth of plant material, protoplast preparation and transfection'''==
The following plant species - ''Arabidopsis thaliana, Brassica rapa, Nicotiana benthamiana'' and ''Hordeum vulgare'' were both germinated and grown in the potting medium under the specific and well controlled circumstances. Photoperiod lasted for 16 horus under the temperature of 22◦C and recorder light intesity was aproximately 160 mol/m2/s [3]. When it comes to the protoplast preparation, they were diluted to 105 ml for process of transfection. Cai et al. followed the protocol written by Yoo et al. which included mixing purified plasmid DNA (4.5 g) and calibrating plasmid together in 96 deep-well (2.2 ml) which contains protoplasts (0.2 ml) [4]. Later, PEG (polyethylene glycol) solution was added to the well, to increase the reacton rate and the overall yield, by mebing a macromolecular crowding agent. They sucessfully produced the transgenic Arabidopsis lines by Agrobacterium mediated tissue transformations and all assembled plasmids were sucessfully transformed into Agrobacterium tumefaciens (they collected the cells by centrifugations and resuspended them into 5% sucrose and seeds from mature siliques). In order to achieve the transgenic lines, all seeds were sterilized with the 70% ethanol for 8-10 minutes and grown on Murashige and Skoog medium [3].

=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==

Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al [7]. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression [3].

=='''Results'''==

It is known that genome engineering technology can provide the functions of cis-regulatory elements (CREs) to be dissected and therefore this research tried to describe the analysis, identification and characterization of plant cis-regulatory elements, by revealing their complexity and role in plants regulatory functions. Also, data that is used, has been helpful with predicting the performance of computationally designed minimal synthetic promoters. Therefore, in order to identifiy candidate cis-regulatory elements for use in minimal synthetic constitutive promoters, promoters which have been used for exogenus expressions had been analyzed with promoters from vascular plants included. Promoters from plant-infecting patogens had been included too, such as CaMV35S, Mirabilis Mosaic Virus (MMV) and A. tumefaciens nopaline synthase - AtuNOS. Achieved results had shown that constitutive promoters do indeed have the ability to bind multiple classes of transcription factors. De novo motif identification was performed by using MEME and the presence of a CRE common (C-CRE) was identified, to all pathogen promoters that were included into the studies. Cho et al. also questioned if the positions of the C-CRE's within the promoter is important so the element was relocated to the transcriptional start site and it was reported that the expression was reduced when the element was moved further
in CAMV35S and MMV but no significant impact was reported in the case with AtuNOS, because it is already located distantly to the transcriptional start site. For identification of a functional design for minimal synthetic plant promoters, they had to build and analyze synthetic promoters with a range of level expressions that corespond to the orthogonal transcriptional factors and the primarily design was based on synthetic TALE- responsive elements which consist of 19 bps of random sequence and a second region of variable lenght and a TATA box sequence and a 43 bp minimal core, transcriptional start site included. It was reported that this type of design can be easily used to build promoters with a wide level expression, just by adding different number of binding sites for initial syntetic TALE responsive element. To conclude, promoters from different type of plant pathogens contain a C-CRE which has a significant effect on the expression levels of both natural and synthetic promoters. The utility of minimal synthetic constitutive promoters in genetic circuits was also demonstrated and proven, when Cho et al. constructed a simple multigene constructs where all promoter elements were strictly synthetic and at first, a minimal synthetic constitutive promoters were used just to initiate transcriptional flow by controlling the expression of a otrhogonal transcriptional factos which sucesfully activated the expression of a reporter and indeed, very similiar expression was detected to circuits where the transcriptional factor was controlled by CAMV35S (ised to initiate the process of transcription in transgenic plants). At last, they provided two possible solutions - minimal synthetic constitutive promoters of different strenghts regulated by endogenous transcriptional factos or minimal synthetic constitutive promoter regulated by snythetic orthogonal transcriptional factor. It is yet to be investigated and revealed how the properties of the minimal synthetic constitutive promoters can be modulated when they are combined with different sequence elements [1,3].

=='''Resources'''==
# W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
# R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research, 44 str. 746 - 752.
# Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48 str. 11845–11856.
# N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.
# S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.
# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.
# G.C. Allen, M.A. Flores-Vergara., S. Krasynanski., S. Kumar et W.F. Thompson: A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 2006, 1 str. 2320–2325.

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T15:47:13Z

Alminatahirovic: /* Resources */

Povzeto po članku: [https://academic.oup.com/nar/article/48/21/11845/5897334 Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48, str. 11845–11856.]

=='''Introduction'''==
Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses [6].

=='''Identification of candidate transcription factor binding sites (TFBSs)'''==
All expression data for transcription factors encoding genes was obtained from the Expression Atlas [2].

=='''Construction of plasmids'''==
Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site)[4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS
terminator [3].

=='''Growth of plant material, protoplast preparation and transfection'''==
The following plant species - ''Arabidopsis thaliana, Brassica rapa, Nicotiana benthamiana'' and ''Hordeum vulgare'' were both germinated and grown in the potting medium under the specific and well controlled circumstances. Photoperiod lasted for 16 horus under the temperature of 22◦C and recorder light intesity was aproximately 160 mol/m2/s [3]. When it comes to the protoplast preparation, they were diluted to 105 ml for process of transfection. Cai et al. followed the protocol written by Yoo et al. which included mixing purified plasmid DNA (4.5 g) and calibrating plasmid together in 96 deep-well (2.2 ml) which contains protoplasts (0.2 ml) [4]. Later, PEG (polyethylene glycol) solution was added to the well, to increase the reacton rate and the overall yield, by mebing a macromolecular crowding agent. They sucessfully produced the transgenic Arabidopsis lines by Agrobacterium mediated tissue transformations and all assembled plasmids were sucessfully transformed into Agrobacterium tumefaciens (they collected the cells by centrifugations and resuspended them into 5% sucrose and seeds from mature siliques). In order to achieve the transgenic lines, all seeds were sterilized with the 70% ethanol for 8-10 minutes and grown on Murashige and Skoog medium [3].

=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==

Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression [3].

=='''Results'''==

It is known that genome engineering technology can provide the functions of cis-regulatory elements (CREs) to be dissected and therefore this research tried to describe the analysis, identification and characterization of plant cis-regulatory elements, by revealing their complexity and role in plants regulatory functions. Also, data that is used, has been helpful with predicting the performance of computationally designed minimal synthetic promoters. Therefore, in order to identifiy candidate cis-regulatory elements for use in minimal synthetic constitutive promoters, promoters which have been used for exogenus expressions had been analyzed with promoters from vascular plants included. Promoters from plant-infecting patogens had been included too, such as CaMV35S, Mirabilis Mosaic Virus (MMV) and A. tumefaciens nopaline synthase - AtuNOS. Achieved results had shown that constitutive promoters do indeed have the ability to bind multiple classes of transcription factors. De novo motif identification was performed by using MEME and the presence of a CRE common (C-CRE) was identified, to all pathogen promoters that were included into the studies. Cho et al. also questioned if the positions of the C-CRE's within the promoter is important so the element was relocated to the transcriptional start site and it was reported that the expression was reduced when the element was moved further
in CAMV35S and MMV but no significant impact was reported in the case with AtuNOS, because it is already located distantly to the transcriptional start site. For identification of a functional design for minimal synthetic plant promoters, they had to build and analyze synthetic promoters with a range of level expressions that corespond to the orthogonal transcriptional factors and the primarily design was based on synthetic TALE- responsive elements which consist of 19 bps of random sequence and a second region of variable lenght and a TATA box sequence and a 43 bp minimal core, transcriptional start site included. It was reported that this type of design can be easily used to build promoters with a wide level expression, just by adding different number of binding sites for initial syntetic TALE responsive element. To conclude, promoters from different type of plant pathogens contain a C-CRE which has a significant effect on the expression levels of both natural and synthetic promoters. The utility of minimal synthetic constitutive promoters in genetic circuits was also demonstrated and proven, when Cho et al. constructed a simple multigene constructs where all promoter elements were strictly synthetic and at first, a minimal synthetic constitutive promoters were used just to initiate transcriptional flow by controlling the expression of a otrhogonal transcriptional factos which sucesfully activated the expression of a reporter and indeed, very similiar expression was detected to circuits where the transcriptional factor was controlled by CAMV35S (ised to initiate the process of transcription in transgenic plants). At last, they provided two possible solutions - minimal synthetic constitutive promoters of different strenghts regulated by endogenous transcriptional factos or minimal synthetic constitutive promoter regulated by snythetic orthogonal transcriptional factor. It is yet to be investigated and revealed how the properties of the minimal synthetic constitutive promoters can be modulated when they are combined with different sequence elements [1,3].

=='''Resources'''==
# W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
# R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research, 44 str. 746 - 752.
# Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48 str. 11845–11856.
# N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.
# S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.
# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.
# G.C. Allen, M.A. Flores-Vergara., S. Krasynanski., S. Kumar et W.F. Thompson: A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 2006, 1 str. 2320–2325.

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T15:45:17Z

Alminatahirovic: /* Results */

Povzeto po članku: [https://academic.oup.com/nar/article/48/21/11845/5897334 Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48, str. 11845–11856.]

=='''Introduction'''==
Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses [6].

=='''Identification of candidate transcription factor binding sites (TFBSs)'''==
All expression data for transcription factors encoding genes was obtained from the Expression Atlas [2].

=='''Construction of plasmids'''==
Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site)[4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS
terminator [3].

=='''Growth of plant material, protoplast preparation and transfection'''==
The following plant species - ''Arabidopsis thaliana, Brassica rapa, Nicotiana benthamiana'' and ''Hordeum vulgare'' were both germinated and grown in the potting medium under the specific and well controlled circumstances. Photoperiod lasted for 16 horus under the temperature of 22◦C and recorder light intesity was aproximately 160 mol/m2/s [3]. When it comes to the protoplast preparation, they were diluted to 105 ml for process of transfection. Cai et al. followed the protocol written by Yoo et al. which included mixing purified plasmid DNA (4.5 g) and calibrating plasmid together in 96 deep-well (2.2 ml) which contains protoplasts (0.2 ml) [4]. Later, PEG (polyethylene glycol) solution was added to the well, to increase the reacton rate and the overall yield, by mebing a macromolecular crowding agent. They sucessfully produced the transgenic Arabidopsis lines by Agrobacterium mediated tissue transformations and all assembled plasmids were sucessfully transformed into Agrobacterium tumefaciens (they collected the cells by centrifugations and resuspended them into 5% sucrose and seeds from mature siliques). In order to achieve the transgenic lines, all seeds were sterilized with the 70% ethanol for 8-10 minutes and grown on Murashige and Skoog medium [3].

=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==

Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression [3].

=='''Results'''==

It is known that genome engineering technology can provide the functions of cis-regulatory elements (CREs) to be dissected and therefore this research tried to describe the analysis, identification and characterization of plant cis-regulatory elements, by revealing their complexity and role in plants regulatory functions. Also, data that is used, has been helpful with predicting the performance of computationally designed minimal synthetic promoters. Therefore, in order to identifiy candidate cis-regulatory elements for use in minimal synthetic constitutive promoters, promoters which have been used for exogenus expressions had been analyzed with promoters from vascular plants included. Promoters from plant-infecting patogens had been included too, such as CaMV35S, Mirabilis Mosaic Virus (MMV) and A. tumefaciens nopaline synthase - AtuNOS. Achieved results had shown that constitutive promoters do indeed have the ability to bind multiple classes of transcription factors. De novo motif identification was performed by using MEME and the presence of a CRE common (C-CRE) was identified, to all pathogen promoters that were included into the studies. Cho et al. also questioned if the positions of the C-CRE's within the promoter is important so the element was relocated to the transcriptional start site and it was reported that the expression was reduced when the element was moved further
in CAMV35S and MMV but no significant impact was reported in the case with AtuNOS, because it is already located distantly to the transcriptional start site. For identification of a functional design for minimal synthetic plant promoters, they had to build and analyze synthetic promoters with a range of level expressions that corespond to the orthogonal transcriptional factors and the primarily design was based on synthetic TALE- responsive elements which consist of 19 bps of random sequence and a second region of variable lenght and a TATA box sequence and a 43 bp minimal core, transcriptional start site included. It was reported that this type of design can be easily used to build promoters with a wide level expression, just by adding different number of binding sites for initial syntetic TALE responsive element. To conclude, promoters from different type of plant pathogens contain a C-CRE which has a significant effect on the expression levels of both natural and synthetic promoters. The utility of minimal synthetic constitutive promoters in genetic circuits was also demonstrated and proven, when Cho et al. constructed a simple multigene constructs where all promoter elements were strictly synthetic and at first, a minimal synthetic constitutive promoters were used just to initiate transcriptional flow by controlling the expression of a otrhogonal transcriptional factos which sucesfully activated the expression of a reporter and indeed, very similiar expression was detected to circuits where the transcriptional factor was controlled by CAMV35S (ised to initiate the process of transcription in transgenic plants). At last, they provided two possible solutions - minimal synthetic constitutive promoters of different strenghts regulated by endogenous transcriptional factos or minimal synthetic constitutive promoter regulated by snythetic orthogonal transcriptional factor. It is yet to be investigated and revealed how the properties of the minimal synthetic constitutive promoters can be modulated when they are combined with different sequence elements [1,3].

=='''Resources'''==
# W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
# R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research, 44 str. 746 - 752.
# Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48 str. 11845–11856.
# N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.
# S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.
# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T15:44:43Z

Alminatahirovic: /* Results */

Povzeto po članku: [https://academic.oup.com/nar/article/48/21/11845/5897334 Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48, str. 11845–11856.]

=='''Introduction'''==
Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses [6].

=='''Identification of candidate transcription factor binding sites (TFBSs)'''==
All expression data for transcription factors encoding genes was obtained from the Expression Atlas [2].

=='''Construction of plasmids'''==
Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site)[4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS
terminator [3].

=='''Growth of plant material, protoplast preparation and transfection'''==
The following plant species - ''Arabidopsis thaliana, Brassica rapa, Nicotiana benthamiana'' and ''Hordeum vulgare'' were both germinated and grown in the potting medium under the specific and well controlled circumstances. Photoperiod lasted for 16 horus under the temperature of 22◦C and recorder light intesity was aproximately 160 mol/m2/s [3]. When it comes to the protoplast preparation, they were diluted to 105 ml for process of transfection. Cai et al. followed the protocol written by Yoo et al. which included mixing purified plasmid DNA (4.5 g) and calibrating plasmid together in 96 deep-well (2.2 ml) which contains protoplasts (0.2 ml) [4]. Later, PEG (polyethylene glycol) solution was added to the well, to increase the reacton rate and the overall yield, by mebing a macromolecular crowding agent. They sucessfully produced the transgenic Arabidopsis lines by Agrobacterium mediated tissue transformations and all assembled plasmids were sucessfully transformed into Agrobacterium tumefaciens (they collected the cells by centrifugations and resuspended them into 5% sucrose and seeds from mature siliques). In order to achieve the transgenic lines, all seeds were sterilized with the 70% ethanol for 8-10 minutes and grown on Murashige and Skoog medium [3].

=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==

Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression [3].

=='''Results'''==

It is known that genome engineering technology can provide the functions of cis-regulatory elements (CREs) to be dissected and therefore this research tried to describe the analysis, identification and characterization of plant cis-regulatory elements, by revealing their complexity and role in plants regulatory functions. Also, data that is used, has been helpful with predicting the performance of computationally designed minimal synthetic promoters. Therefore, in order to identifiy candidate cis-regulatory elements for use in minimal synthetic constitutive promoters, promoters which have been used for exogenus expressions had been analyzed with promoters from vascular plants included. Promoters from plant-infecting patogens had been included too, such as CaMV35S, Mirabilis Mosaic Virus (MMV) and A. tumefaciens nopaline synthase - AtuNOS. Achieved results had shown that constitutive promoters do indeed have the ability to bind multiple classes of transcription factors. De novo motif identification was performed by using MEME and the presence of a CRE common (C-CRE) was identified, to all pathogen promoters that were included into the studies. Cho et al. also questioned if the positions of the C-CRE's within the promoter is important so the element was relocated to the transcriptional start site and it was reported that the expression was reduced when the element was moved further
in CAMV35S and MMV but no significant impact was reported in the case with AtuNOS, because it is already located distantly to the transcriptional start site. For identification of a functional design for minimal synthetic plant promoters, they had to build and analyze synthetic promoters with a range of level expressions that corespond to the orthogonal transcriptional factors and the primarily design was based on synthetic TALE- responsive elements which consist of 19 bps of random sequence and a second region of variable lenght and a TATA box sequence and a 43 bp minimal core, transcriptional start site included. It was reported that this type of design can be easily used to build promoters with a wide level expression, just by adding different number of binding sites for initial syntetic TALE responsive element. To conclude, promoters from different type of plant pathogens contain a C-CRE which has a significant effect on the expression levels of both natural and synthetic promoters. The utility of minimal synthetic constitutive promoters in genetic circuits was also demonstrated and proven, when Cho et al. constructed a simple multigene constructs where all promoter elements were strictly synthetic and at first, a minimal synthetic constitutive promoters were used just to initiate transcriptional flow by controlling the expression of a otrhogonal transcriptional factos which sucesfully activated the expression of a reporter and indeed, very similiar expression was detected to circuits where the transcriptional factor was controlled by CAMV35S (ised to initiate the process of transcription in transgenic plants). At last, they provided two possible solutions - minimal synthetic constitutive promoters of different strenghts regulated by endogenous transcriptional factos or minimal synthetic constitutive promoter regulated by snythetic orthogonal transcriptional factor. It is yet to be investigated and revealed how the properties of the minimal synthetic constitutive promoters can be modulated when they are combined with different sequence elements

=='''Resources'''==
# W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
# R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research, 44 str. 746 - 752.
# Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48 str. 11845–11856.
# N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.
# S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.
# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T15:29:47Z

Alminatahirovic: /* Results */

Povzeto po članku: [https://academic.oup.com/nar/article/48/21/11845/5897334 Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48, str. 11845–11856.]

=='''Introduction'''==
Both synthetic promoters and transcription factors (TFs) have been powerful components, responsible for precise and exact regulation of specific plant transgene expression. For rational design of minimal synthetic plant promoters, we require specific type, spacing of motifs placed upstream of synthetic promoter and a copy of number. Synthetic transcription factors can be similarly constructed with usage of variety of DNA binding domains (DBs) and efffector domains as well. What makes synthetic promoters and transcription factos better and more efficient than their natural counterparts is ability to provide better and more adequate transgene expression strenght and specificity [1]. Synthetic promotors consist of a core promotre and synthetic motifs which are primarly focused on control of transgene expression, with motif sequence being derived from an extant sequence or multiplied. The core promoter contains a TATA-box and GA elements and the best known plant core promoter is the minimal CaMV 35S promoter. Scientists are trying to produce synthetic core promoters by using different TATA-box regions and core elements, even though they can be identified from native plant genes and common viruses [6].

=='''Identification of candidate transcription factor binding sites (TFBSs)'''==
All expression data for transcription factors encoding genes was obtained from the Expression Atlas [2].

=='''Construction of plasmids'''==
Constructs were designed by using Benchling (modern software platforme used to programmatically access and edit data) and multigene constructs were sucessfully assembled with the Type IIS DNA assembly protocol - type IIS restriction enzymes recognize nonpalindromic sequence motifs and cleave outside of their recognition site)[4]. In order to achieve more stable plant transformation, both synthetic and control promoters were sucessfully assembled with the following: a) 5'UTR from cowpea mosaic virus; b) chimeric coding sequence consisting of an N -terminal HiBit; c) a C-terminal yellow fluorescent protein and d) AtuOCS
terminator [3].

=='''Growth of plant material, protoplast preparation and transfection'''==
The following plant species - ''Arabidopsis thaliana, Brassica rapa, Nicotiana benthamiana'' and ''Hordeum vulgare'' were both germinated and grown in the potting medium under the specific and well controlled circumstances. Photoperiod lasted for 16 horus under the temperature of 22◦C and recorder light intesity was aproximately 160 mol/m2/s [3]. When it comes to the protoplast preparation, they were diluted to 105 ml for process of transfection. Cai et al. followed the protocol written by Yoo et al. which included mixing purified plasmid DNA (4.5 g) and calibrating plasmid together in 96 deep-well (2.2 ml) which contains protoplasts (0.2 ml) [4]. Later, PEG (polyethylene glycol) solution was added to the well, to increase the reacton rate and the overall yield, by mebing a macromolecular crowding agent. They sucessfully produced the transgenic Arabidopsis lines by Agrobacterium mediated tissue transformations and all assembled plasmids were sucessfully transformed into Agrobacterium tumefaciens (they collected the cells by centrifugations and resuspended them into 5% sucrose and seeds from mature siliques). In order to achieve the transgenic lines, all seeds were sterilized with the 70% ethanol for 8-10 minutes and grown on Murashige and Skoog medium [3].

=='''Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression'''==

Cho et al. sucessfully extracted the DNA using the cetyltrimethylammonium bromide extraction protocol described by Allen et al. After digesting a 2mg of genomic DNA with 20 units EcoRV for aproximately 2 hours on the temperature of 37◦C, they used the 400 ng of total digested genomic DNA in digital droplet polymerase chain reaction (ddPCR) along with oligonucleotide primers to the transgene sequence (5' -CGGCGAAATTCCAT ACCTGTT and 5' -TCAGCCGATTATCATCACCGA). Nano-Glo Dual-Luciferase reporter assay system was used to detect the luciferase expression [3].

=='''Results'''==

It is known that genome engineering technology can provide the functions of cis-regulatory elements (CREs) to be dissected and therefore this research tried to describe the analysis, identification and characterization of plant cis-regulatory elements, by revealing their complexity and role in plants regulatory functions. Also, data that is used, has been helpful with predicting the performance of computationally designed minimal synthetic promoters. Therefore, in order to identifiy candidate cis-regulatory elements for use in minimal synthetic constitutive promoters, promoters which have been used for exogenus expressions had been analyzed with promoters from vascular plants included. Promoters from plant-infecting patogens had been included too, such as CaMV35S, Mirabilis Mosaic Virus (MMV) and A. tumefaciens nopaline synthase - AtuNOS. Achieved results had shown that constitutive promoters do indeed have the ability to bind multiple classes of transcription factors. De novo motif identification was performed by using MEME and the presence of a CRE common (C-CRE) was identified, to all pathogen promoters that were included into the studies. Cho et al. also questioned if the positions of the C-CRE's within the promoter is important so the element was relocated to the transcriptional start site and it was reported that the expression was reduced when the element was moved further
in CAMV35S and MMV but no significant impact was reported in the case with AtuNOS, because it is already located distantly to the transcriptional start site. For identification of a functional design for minimal synthetic plant promoters, they had to build and analyze synthetic promoters with a range of level expressions that corespond to the orthogonal transcriptional factors and the primarily design was based on synthetic TALE- responsive elements which consist of 19 bps of random sequence and a second region of variable lenght and a TATA box sequence and a 43 bp minimal core, transcriptional start site included. It was reported that this type of design can be easily used to build promoters with a wide level expression, just by adding different number of binding sites for initial syntetic TALE responsive element.

=='''Resources'''==
# W.Liu., C.N. Stewart Jr: Plant synthetic promoters and transcription factors. Current Opinion in Biotechnology. 2016, 37 str. 36–44.
# R.Petryszak.,M.Keays., Y.A.Tang., N.A. Fonseca., E. Barrera., T. Burdett, A.M.P. Fuentes., S. Jupp., S. Koskinen et al. Expression Atlas update––an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Research, 44 str. 746 - 752.
# Y.M. Cai., K. Kallam., H.Tidd., G. Gendarini., A. Salzman et N. J. Patron: Rational design of minimal synthetic promoters for plants. Nucleid Acids Research. 2020, 48 str. 11845–11856.
# N.J.Patron., D. Orzaez., S. Marillonnet et al: Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist Foundation. 2015, 208 str. 13 - 19.
# S.D. Yoo., Y.H. Cho et J.Sheen: Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocol. 2007, 2 str. 1565–1572.
# T.Vogl, C.Ruth, J. Pitzer, T. Kickenweiz, A. Glieder: Synthetic core promoters for ''Pichia pastoris''. ACS Synthetic Biology. 2014, 3 str. 188-191.

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T15:14:19Z

Alminatahirovic: /* Determination of transgene copy number by digital droplet PCR (ddPCR) and quantification of gene expression */

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T15:02:44Z

Alminatahirovic: /* Growth of plant material, protoplast preparation and transfection */

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T14:49:40Z

Alminatahirovic: /* Resources */

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T14:49:11Z

Alminatahirovic: /* Introduction */

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T14:48:28Z

Alminatahirovic: /* Resources */

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T14:41:57Z

Alminatahirovic: /* Introduction */

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T14:35:30Z

Alminatahirovic: /* Growth of plant material, protoplast preparation and transfection */

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T14:31:15Z

Alminatahirovic: /* Growth of plant material, protoplast preparation and transfection */

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T14:30:34Z

Alminatahirovic: /* Growth of plant material, protoplast preparation and transfection */

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T14:14:50Z

Alminatahirovic: /* Resources */

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T14:13:21Z

Alminatahirovic: /* Construction of plasmids */

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T14:04:57Z

Alminatahirovic: /* Resources */

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T14:02:55Z

Alminatahirovic: /* Construction of plasmids */

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T13:59:02Z

Alminatahirovic: /* Resources */

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T13:58:41Z

Alminatahirovic: /* Construction of plasmids */

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T13:57:56Z

Alminatahirovic: /* Identification of candidate transcription factor binding sites (TFBSs) */

Racionalna zasnova minimalnih sintetičnih promotorjev za rastline

2021-04-19T13:38:53Z

Alminatahirovic: /* Resources */