Difference between revisions of "Team:Toulouse France/Description"

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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