Team:Paris Bettencourt/Human Practice/Safety


Safety forms were approved on October 3, 2013 by Evan Appleton.

Safety and health of team members or others working in the lab

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.

Eliminating Mycobacteria: This project employs the Listeriolysin gene LLO derived from Listeria monocytogenes. This gene contributes the virulence of this pathogen. Although this gene is widely used in Biosafety Level 1 facilities, the precautionary principle applies. We will assume that this gene could enhance the pathogenicity of E. coli or other bacteria, for example by allowing them to evade phagocytosis in the human immune system.

Screening for sulfur metabolism inhibitors: For this project we will express in E. coli three genes from the sulfur assimilation pathway of Mycobacterium smegmatis. We selected genes from this organism because it is a non-pathogenic model for M. tuberculosis. To our knowledge, sulfur metabolism does not enhance the pathogenicity of any known bacterial species. Therefore we believe this project to carry risks not exceeding those of standard lab work with E. coli.

Risks to the safety and health of the general public, if released by design or by accident

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.

Detecting antibiotic resistance: As above, this project carries the risks inherrent to basic M13 phage work.

Screening for sulfur metabolism inhibitors: We intend to replate WT E. coli sulfur metabolism, e.g. CysI, with alternative genes from M. smegmatis such as SirA. The SirA gene is well characterized and unlikely to relate to pathogenicity. Because both these organisms belong to Risk Group 1, we believe there is little risk to the public.

Risks to the environment, if released by design or by accident

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.

Alterations of E. coli sulfur metabolism, if released, could potentially disrupt the sulfur cycle in natural bacterial ecosystems. For this to occur, our bacteria would not only need to escape routine laboratory containment practices, but also become established in a natural niche. We consider it highly unlikely that our modified bacterial strains could compete with natural strains in their native environments.

Risks to security through malicious misuse by individuals, groups, or countries

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,

this construct could in principle be used to maliciously enhance bacterial pathogenicity. To our knowledge, Listeria pathogenicity is not yet sufficiently understood that it could be intentionally reproduced in other species. Nevertheless, we take the biosafety concerns of this construct very seriously and we discuss them further in the text below. Sulfur metabolism is unrelated to pathogenicity, and we do not think of it could be maliciously misused in any way.

Risks that might arise if the project would be used as a commercial/industrial product

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.

Eliminating Mycobacteria: As above, this system is designed to be used outside the clinic. Further, it is imaging to be a medicine inside the human body. Therefore, in addition to the complexities discussed above, this project must be proven safe for use in human subjects. We don't think that this will be easy, but we do think that the potential public health benefits of this project could some day justify the difficulty.

Screening for sulfur metabolism inhibitors: For this project, all the synthetic biology will be done in the lab. We intend to produce a small-molecule therapeutic identified by our drug screen. Small molecule drugs require extensive testing before being approved for human use, but the regulatory framework for their approval is well-established.

Safety training

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.

Rules about lab protocol, protective clothing and equipment, decontamination methods and practices, prevention of the transfer of genetic material and microorganisms, handling of chemicals, machines and emergency procedures.

In addition to the BSL 1 safety training, team members working with Risk Group 2 parts received BSL 2 safety training. It was conducted by the INSERM U1001 group personnel responsible for BSL 2 laboratory and it included additional safety precautions and procedures specific for the BSL 2: Protective clothing and equipment, decontamination practices, prevention of the transfer of genetic material and microorganisms, emergency procedures, use and handling of machines, etc. Also, they were explained the design feautures unique to a BSL 2 lab.

Institutional Biosafety Committee and changes made to the project based on their review

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

2012 iGEM team, winner of the Safety Commendation and Best Environment Project [bWARE: How safe is safe enough?].

During the selection of our project, many ideas were considered, and some raised safety issues. Especially while thinking about the tuberculosis, we knew there were serious risks to be considered because of its pathogenic nature. After conversations with several senior researchers in our lab, reading the iGEM safety guidelines and following experience from previous iGEM teams we decided not to use genes from Mycobacterium tuberculosis but rather non-­infectious host organism like M. smegmatis to serve as a proof-of-principle.

The BioSafety Level rating of our lab according to the WHO Biosafety Manual

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.

The Risk Group of our parts and chassis organisms

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.

Risk Group 2 part - Listeriolysin O

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.

We don't believe that hly gene isn't dangerous - hly carring strain should be able to invade any eucaryotic cell performing phagocytosis of extracellular bacteria. As mentioned above, special safety measures have been taken in account while working with bacteria transformed with this part.

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
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