Team:ULB-Brussels/Safety

$\Tgo$he main concerns raised by $\MyColi$ depend more of the protein that we chose to produce than of itself. However, $\MyColi$ could compel an escaped recombinant bacterium to produce an industrial protein in the environment, when a bacterium without our system would quickly degenerate to stop producing the protein of industrial interest. The risk seems thin, since such an overproducing bacterium would suffer from a clear competitive disadvantage in a wild environment, but it is not excluded that the plasmid containing the $\MyColi$ system maintains itself anyway through Horizontal Gene transfer (HGT), at the expend of the wild bacteria it infects, benefiting of the proprieties of the TA system.

In the current state of our project, the protein production is boosted thanks to a TA system, but the plasmids are maintained through the usual system of antibiotic resistance, since the toxin and the antitoxin will be placed on different plasmids bearing different resistance genes. The system will thus decay quickly if $\MyColi$ bacteria wander in a wild environment, without any antibiotic. In the final version of our project (that is, if we have the time to go this far), the toxin gene will be inserted in the genomic DNA of the bacteria, preventing it to ever be lost, and compelling the bacteria to keep the plasmid bearing the antitoxin and the protein of interest genes. It will also prevent any reasonable chance of Horizontal Gene Transfer of the $\MyColi$ System.

If, for one reason or another, the overproduction of a protein did not results in a competitive disadvantage, it would be wise to implement a build-in suicide sequence in the bacteria, to prevent it surviving outside the bioreactor. It can easily be done by the subordination of the antitoxin production to an inducible promoter that would be activated by a compound only present in the bioreactor. Several of containment devices have been developed by iGEM teams and other are already in use in the industry.

In conclusion, $\MyColi$ seems to be an extremely safe and predictable system, whose the biggest danger would be to be used to produce a dangerous protein. This latter problem has to be addressed on a case-by-case basis, by other institution than ours. However, our topical $\MyColi$ lab system isn't dangerous, producting global fluorescent proteins (GFP or RFP).