Team:Paris Bettencourt/Human Practice/Safety

Silencing antibiotic resistance: This system includes a phage designed to spread in an E. coli population. If it was ingested by someone the phage could spread into the population of E. coli located in the gut. The modifications to our phage, for example GFP expression, are likely to reduce fitness and would facilitate diagnosis. We believe this risk to be comparable to that of other research on commonly used coliphage.

Detecting antibiotic resistance: As above, this system uses a coliphage that could in principle spread in human gut fauna. This system also uses CRISPR elements, which are relatively new to science and may present unknown risks. We do not believe that our modifications will increase the risks above that of other research on commonly used coliphage.

Silencing antibiotic resistance: The M13 bacteriophage that we use in this project may be capable of spreading in human gut flora. We do not believe that the risks exceed that of standard coliphage research.

Eliminating Mycobacteria: Listeria monocytogenes is a human pathogen, and the LLO gene that we employ contributes to that pathogenicity. Although the LLO gene is only one of many genes required for Listeria pathogenicity, we must assume there is some chance it could enhance the pathogenicity of other strains. Release of this strain could, in principle, enhance the pathogenicity of natural human pathogens, particularly that of E. coli and it's relatives.

In principle, a released coliphage could disrupt natural bacterial ecosystems, for example in waste water. We do not believe that our projects employing the M13 bacteriophage pose any risks to the environment beyond those of common bacteriophage research. The LLO gene, as a pathogenicity-related factor, poses no specific environmental risks that we are aware of.

The M13 phage vectors that we use could, in principle, spread in human gut flora. Therefore the M13 phage could be maliciously used to spread pathogenic or otherwise harmful genes. We do not see any risks in this category specific to our use of this vector beyond the risks of standard bacteriophage research. The LLO gene contributes to Listeria pathogenicity. If used in combination with multiple other pathogenicity elements,

Silencing antibiotic resistance: This phage-based system is designed to be used outside the clinic. Our concept is that it would spread in a natural Mycobacterial population and counter select antibiotic-resistant genotypes. Applications of this type are unprecedented, and as far as we know there is no regulatory framework for safely engineering global pathogen populations with a free-circulating phage. Therefore, this project must remain in the proof-of-concept phase and can not be scaled up until such a regulatory framework exists. We want to contribute to the development of this framework, but it is beyond the scope of one iGEM project.

Detecting antibiotic resistance: This project is designed for use only in the clinic, and could therefore benefit from the full measures of clinical containment. Because we believe that the phage that we produce will also be harmless, we are confident that our phage sensor could be scaled up without risk.

At the beginning of the competition all of the team members received safety training for working in the Biosafety Level 1 laboratory. The team members that are working with parts derived from Risk Group 2 organisms received safety training for working in the Biosafety Level 2 laboratory. Only those team members were allowed to use the BSL 2 laboratory. Moreover, the Biosafety officer of our laboratory followed our work and students working in the BSL 2 laboratory were supervised by the responsible staff members.

The safety training was conducted by the Biosafety officer for INSERM U1001 and it included best laboratory practices as described in the WHO laboratory biosafety manual.

The work was carried out in the laboratory of Evolutionary Systems Biology at the Molecular, Evolutive and Medical Genetics Unit (U1001, also known as TaMaRa's lab) of the French National Institute of Medical Research (INSERM) within the Paris Descartes University's Medical faculty. More importantly, the biosafety officer of our unit supervised our work.

Both institutions have ethical committees though no specific issue concerning our project was raised. Nonetheless, a thorough work was done throughout this period regarding general safety and ethical issues following the process we established in 2009 iGEM team, winner of the Human Practice Award [Ethics and Biosafety] and

The BioSafety Level rating of the lab all the team members have access to is BSL 1. To access the BSL 2 lab, team members who needed access got a special BSL 2 safety training. BSL 2 work was carried out only under the supervision of a senior lab manager.

All the parts and organisms we are using are Risk Group 1 except the Listeriolysin O gene that comes from a Risk Group 2 parent organism. All the parts and organisms do match the BSL rating of our laboratories.

Hly gene is encoding Listeriolysin O (LLO) lacking its secretion signal sequence. LLO is a hemolysin derived from L. monocytogenes which enables phagosomal membrane lysis. We use hly carrying E. coli to deliver TDMH protein to the cytosol of the macrophages. According to our literature search, there isn't any better part that could fulfill this purpose. E. coli BL21 (DE3) expressing listeriolysin may be able to invade any eucaryotic cell, although literature data suggest that it requires other proteins like invasin or internalin.