Difference between revisions of "Team:Toulouse France/Description"
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<br><br><b style="font-size:16px;">Epsilon/Zeta toxin-antitoxin couple</b> | <br><br><b style="font-size:16px;">Epsilon/Zeta toxin-antitoxin couple</b> | ||
| − | <br><br>The Epsilon and Zeta Toxin-antitoxin system was discovered in Streptococcus pyogenes more precisely in the low-copy-number plasmid pSM19035, its function is to stabilize plasmid segregation. The zeta toxin has shown toxic effects on Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae | + | <br><br>The Epsilon and Zeta Toxin-antitoxin system was discovered in Streptococcus pyogenes more precisely in the low-copy-number plasmid pSM19035, its function is to stabilize plasmid segregation. The zeta toxin has shown toxic effects on Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae (Zielenkiewicz and Cegłowski, 2005). The zeta toxins phosphorylate the peptidoglycane precursor that inhibits the bacterial peptidoglycane synthesis (Mutschler et al., 2011). Without abilities to cell wall synthesis bacteria are lyses. |
<br><br><b style="font-size:16px;">Cloning approach:</b> | <br><br><b style="font-size:16px;">Cloning approach:</b> | ||
| − | <br><br>We synthesized our constructions thanks to IDT gblock. Each fragment contains a toxin but not its corresponding antitoxin. Therefore building our plasmids needed a strategy to avoid cell death. We chose to use theophylline sensitive riboswitch. In the absence of the ligand RBS sequence is available and RNA translation is possible. In ligand presence RNA shape change and RBS is not | + | <br><br>We synthesized our constructions thanks to IDT gblock. Each fragment contains a toxin but not its corresponding antitoxin. Therefore building our plasmids needed a strategy to avoid cell death. We chose to use theophylline sensitive riboswitch. In the absence of the ligand RBS sequence is available and RNA translation is possible. In ligand presence RNA shape change and RBS is not accessible (Topp and Gallivan, 2008). In this case theophylline riboswitch is among our toxin sequence. Theophylline presence represses the toxin expression. These conditions allow us to transform our bacteria with a single plasmid in a media containing 1mM Theophylline. |
Revision as of 09:19, 11 October 2016
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How to help Lascaux ?
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Introduction
Just like in many countries, prehistorical men walked into our lands.
However, they left a trace of their passage in France: the Lascaux cave, which is one of the most important heritages we have.
What is so specific about this cave ?
it is not just a simple cave, cold, rocky and moist. The Lascaux cave is so much more.
Decorated with organic and minerals pigments, it is home to parietal paintings and many frescoes and engravings cover the walls.
Bulls, aurochs and horses are part of these representations, and testify the way of life of our ancestors.
Background - the historic of the cave
As soon as the cave was discovered in 1940, people came from the whole country and abroad to visit it. However, the carbon dioxide and the water vapour released by their breath started to disrupt the cave’s ecosystem. The damages caused by these air changements are at the origin of the first crisis that the Lascaux cave knew. Two kinds of contaminations were observed: the limestone formation on the walls, which was called the white disease, and the green algae growth, named the green disease.
Then, white mold and black stains appeared on the walls and the floor, and were characteristical of the second and the third crisis respectively. These contaminations flow from fungi and bacteria proliferation.
Treatment of the cave
Curing the cave was a priority. Consequently, treatments like biocid or antibiotics were tried.
The success of these initiatives worked temporarily, but new microorganisms developed on the organic wastes.
According to the last reviews about the Lascaux cave, bacteria (Bacillus subtilis, Pseudomonas species, Rhizobium radiobacter,...) and fungi (Fusarium sp., Ochroconis anomalaand, Aspergillus sp., Acremonium sp.,...) are currently in the cave.
Nowadays, even if the cave situation is steady, microorganisms covering frescoes are still present.
Why did we choose this subject?
At the beginning of the iGEM adventure, almost twenty subjects were considered.
Every thematic was intertwined with the other, like environment, health, and mostly art.
After months of intense brainstorming, we finally decided to work on the conservation of the Lascaux cave.
We wanted to choose a project that was personal and original.
The Lascaux cave then imposed itself.
It is part of the French heritage, and was added to the UNESCO World Heritage Sites list.
It has been threatened of destruction since decades now, and organizations from all around the world have gathered to solve the Lascaux cave crisis.
The Lascaux cave is what traces us back to our humanity.
The first men lived there and some of the first pieces of art ever were created there.
Introduction
The historic of the cave
Treatment of the cave
Why did we choose this subject?
Containment:
Ours project involved release of genetically modified bacteria in the cave. Lascaux cave is not a completely closed system; there is interaction with external environment due to water infiltration. The threat of releasing bacteria in the cave is the possibility of horizontal gene transfer to native bacteria. The horizontal gene transfer (or lateral gene transfer) is transmission of DNA and it is known to occur between different species. Here the aim of the containment module was to provide transfer of non natural genetic content to endogenous cave bacteria
Paleotilis project involve more than 15 kb of gene therefore a two plasmid construction was necessary. To provide any of these plasmids dissemination a double toxin-antitoxin was designed (Figure1). One plasmid contains antitoxin 1 and toxin 2, the other contains antitoxin 2 and toxin 1. This construction divide toxin-antitoxin couples, the toxin is on a different plasmid then it corresponding antitoxin. Through this system both plasmids are compulsory for bacteria survival. If endogenous bacteria of the cave received a single plasmid, toxin expression will lead to death. In the same time this design obligates the cell to maintain both (or none) plasmids.
The toxin –antitoxin are under the control of a constitutive promoter of Bacillus subtilis: Pveg (BBa_K143012), to assure a constant expression of the toxin (Figure2). The two pairs of Toxin-Antitoxin combinations selected for this project are MazF/MazE and Zeta/Epsilon. Both are type II Toxin-Antitoxin systems, meaning that antitoxin and toxin are proteins and the protein toxin is inhibited by the binding of a protein antitoxin (Wang et al., 2013)
Figure 1: Design of the containment system of paleotilis
MazF /MazE toxin-antitoxin couple
The mazEF operon was the first Toxin-Antitoxin system found on the Escherichia coli chromosome. It is related to the kis/kid module on plasmid R1 (Bravo et al., 1987). MazF is an endoribonuclease which specifically cleaves mRNAs at ACA sequences (Zhang et al., 2005). In Escherichia coli mazF inhibits protein synthesis by cleaving mRNAs. The excess of MazF toxin (in case of single plasmid) provide by our system will lead to bactericidal effect.
Epsilon/Zeta toxin-antitoxin couple
The Epsilon and Zeta Toxin-antitoxin system was discovered in Streptococcus pyogenes more precisely in the low-copy-number plasmid pSM19035, its function is to stabilize plasmid segregation. The zeta toxin has shown toxic effects on Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae (Zielenkiewicz and Cegłowski, 2005). The zeta toxins phosphorylate the peptidoglycane precursor that inhibits the bacterial peptidoglycane synthesis (Mutschler et al., 2011). Without abilities to cell wall synthesis bacteria are lyses.
Cloning approach:
We synthesized our constructions thanks to IDT gblock. Each fragment contains a toxin but not its corresponding antitoxin. Therefore building our plasmids needed a strategy to avoid cell death. We chose to use theophylline sensitive riboswitch. In the absence of the ligand RBS sequence is available and RNA translation is possible. In ligand presence RNA shape change and RBS is not accessible (Topp and Gallivan, 2008). In this case theophylline riboswitch is among our toxin sequence. Theophylline presence represses the toxin expression. These conditions allow us to transform our bacteria with a single plasmid in a media containing 1mM Theophylline.
References
Bravo, A., de Torrontegui, G., and Díaz, R. (1987). Identification of components of a new stability system of plasmid R1, ParD, that is close to the origin of replication of this plasmid. Mol. Gen. Genet. MGG 210, 101–110.
Mutschler, H., Gebhardt, M., Shoeman, R.L., and Meinhart, A. (2011). A novel mechanism of programmed cell death in bacteria by toxin-antitoxin systems corrupts peptidoglycan synthesis. PLoS Biol. 9, e1001033.
Topp, S., and Gallivan, J.P. (2008). Riboswitches in unexpected places—A synthetic riboswitch in a protein coding region. RNA 14, 2498–2503.
Wang, X., Lord, D.M., Hong, S.H., Peti, W., Benedik, M.J., Page, R., and Wood, T.K. (2013). Type II Toxin/Antitoxin MqsR/MqsA Controls Type V Toxin/Antitoxin GhoT/GhoS. Environ. Microbiol. 15, 1734–1744.
Zhang, Y., Zhang, J., Hara, H., Kato, I., and Inouye, M. (2005). Insights into the mRNA Cleavage Mechanism by MazF, an mRNA Interferase. J. Biol. Chem. 280, 3143–3150.
Zielenkiewicz, U., and Cegłowski, P. (2005). The Toxin-Antitoxin System of the Streptococcal Plasmid pSM19035. J. Bacteriol. 187, 6094–6105.
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