Team:ULB-Brussels/Safety

The main concerns raised by Mighty Coli depend more on the protein that we chose to produce rather than on Mighty Coli itself. However, Mighty Coli could compel an escaped recombinant bacterium to produce an industrial protein in the environment, when a bacterium without our system would quickly degenerate and stop producing the protein of interest. The risk seems thin, since such an overproducing bacterium would suffer from a clear competitive disadvantage in a wild environment.

However, in the current state of our project, plasmids are maintained in the bacterial population through the usual system of antibiotic resistance, since the toxin and the antitoxin are to be placed on different plasmids bearing different resistance genes. The properties of the TA systems are used to boost protein production but also for plasmid stability. In the final version of our project, the toxin gene will be inserted in the genomic DNA of the bacteria to reduce loss chances. Bacteria will be compelled to keep the plasmid bearing the genes of the antitoxin and the protein of interest, without the need of infamous antibiotic resistance genes

If for one reason or another, the overproduction of a protein didn't result in a competitive disadvantage, antitoxin production can be put under the control of an inducible promoter, acting as a built-in biocontainment device. Indeed, if we chose an artificial inducer for the expression of the antitoxin (that is, an inducer not found in the nature), an escaped bacterium couldn't inhibit the activity of the toxin and woud quickly die outside of the bioreactor.

Rules of good handling should be respected for our system. Theses rules vary to one country to another. According to the Belgian Biosafety Server, Migthy coli is a class 1 risk because our construction cannot cause any disease in animals or plants or cause disorders in the environnement. The risk class depends on the modified organism. To manipulate class 1 organisms, the laboratory must have different features :

• A sink to wash and decontaminate hands
• A cloakroom where lab coats are not in contact with everyday clothing
• Easy to clean benches, resistant to acidic, alkaline and disinfectant solutions
• An autoclave to sterilize waste
• Laboratories restricted to authorized personnel
• Class risk and lab head are indicated on the entrance door of the lab
• Mandatory lab coats to work
• Confinement of modified organisms into a sealed system when they are not used
• Mechanical pipetting appartus
• Prohibiting of eating, drinking, smoking, manipulating contact lens, using cosmetics or stocking food for human consummation in the lab
• A notebook where each modified organism which is used and stocked is recorded
• Enforcement of lab security
• Hands are washed before leaving the lab
• Disinfection of benches after each manipulation and also if biological material is spilled

• A manual of decontamination is at the disposition of the workers
• Courses about security are applied for the workers and regular updated are organized
• Pets are forbidden in the lab
• Waste and contaminated materials are inactivated by an appropriate and validated method (by incineration or autoclave)

In conclusion, our system seems to be extremely safe and predictable. Its biggest danger consists of being used to produce a dangerous protein. This latter problem should be addressed on a case-by-case basis, by other institution than ours. Of course, our topical lab system is really safe, since it should only overproduce fluorescent reporters (GFP or RFP). However, Mighty Coli will be more useful to produce cumbersome proteins than usual fluorescent proteins.