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We present here our reflexion about safety issues of the “Biofilm Killer” project based on a modified Bacillus subtilis strain able to swarm into biofilms, to produce a biocide agent and a dispersive agent. To obtain genetic constructions, we worked with an Escherichia coli strain. Staphylococcus epidermidis, S. aureus and adherent E. coli strains were used as biofilm models.

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Researcher/Public/Environmental Safety

Health and safety training

Before starting experimental work, we have identified chemical/biological Hazards and Risks. We consider that having a proper training in safety and security at the beginning of our experimental work prepared us to be more organized, responsible for our actions and respectful for those of others.
We have followed all instructions from our institution concerning the lab electrical and gas systems. Emergency numbers are displayed near the phones. No one was allowed to work alone in the laboratory. In addition, the French system provides all young adults with a training in first-aid and safety. Moreover, several students, advisors and instructors have the life-saving diploma and are also trained for firefighting. A specific formation for handling chemicals and modified bacteria carrying antibiotic resistance genes was given to each student before starting the bench work.

Restricted access

Laboratory experiments always imply handling hazardous substances. And their use can present a risk for the health of the manipulator or for the environment if not stored, used and eliminated in waste properly, according to their harmfulness. For these reasons, the access of the laboratory was limited to those involved in the project only.
Each room is equipped with labels on each door to inform people of what they may find inside and what safety procedures they need to follow.

Waste management

Chemical wastes. All reagents were eliminated in the appropriate waste recovery barrels.

Biological wastes. We used the autoclave of the microbiology teaching platform to decontaminate our biological solid and liquid wastes : no bacteria, modified or not, are released in the environment.


All work benches were cleaned every day. The whole lab was cleaned every week.


Some of the reagents used are irritant, toxic and can be potential carcinogens (polyacrylamid, Ethidium bromide…). To minimize the impact of their use, they were manipulated following the supplier’s instructions, wearing appropriate personal safety equipment, i.e. gloves, safety glasses, labcoats and under extractor hood when necessary. All samples, tubes, vials were clearly identified/labelled to avoid inappropriate mix between two non-compatible solvents.

We took specific safety measures for the use of Ethidium Bromide (EtBr). Ethidium bromide is known to act as a mutagen because it intercalates within the double strand DNA helix. To avoid the dissemination of EtBr in the lab, it was stored and used in the same room where the electrophoresis gels were revealed. EtBr was NEVER incorporated into the electrophoresis gels, but used in staining bath instead to avoid the contamination of electrophoresis equipment. Specific trash barrel for genotoxic contaminated material were used for EtBr-contaminated material.


Specific biological hazards and risks linked to the “Biofilm Killer” project

Bacterial strains

All Bacillus subtilis, E. coli and S. epidermidis strains we used at INSA-Lyon have a biosafety level of 1, which means that they are not known to cause disease and have minimal environmental hazards. Staphylococcus aureus has a biosafety level of 2 and requires P2 equipment. Manipulations with this organism were conducted in the Bioadhesion/Biofilm lab in Massy (Paris) under Romain Briandet's supervision.

B. subtilis is the chassis chosen for “Biofilm Killer”. B. subtilis is widely used in the field as a biocontrol agent against plant pathogens and food industry processes. It has been granted Qualified Presumption of Safety status by the European Food Safety Authority (EFSA). However, if modified by biobricks, B. subtilis has to be physically contained (double filter 0.45/0.2 µm)( See process in "Our solution")

. Enhanced safety can be achieved by inserting functions in the chassis that prevent horizontal tranfert of DNA from our bacterium to another one. This aspect is developed in the chapter "New ideas for safety in iGEM"

"The Bacillus subtilis species has a long history of safe use. It has been granted Qualified Presumption of Safety (QPS) status by the European Food Safety Authority (EFSA) and is part of the authoritative list of microorganisms with a documented history of safe use in food established by the International Dairy Federation (IDF) in collaboration with the European Food and Feed Cultures Association (EFFCA) in 2002 and updated in 2012." [1] B. subtilis is known to produce endospores [2] that are resistant to different treatments (heat, UV irradiation…) [3]. Spores of a variety of species are of major concern for the food industry, and even if B. subtilis is non-pathogenic, use of a sporulating strain can be a problem in food industry because spores are resistant to most cleaning procedure and thus can get out of usual control. Therefore, we plan to introduce a mutation in the B. subtilis strain 168 to disrupt the sporulation process and offer an asporulating version of Biofilm Killer for food application.

“Biofilm Killer” power: Lysostaphin, Dispersin, Surfactin

None of the parts used raise any specific safety issues. The final engineered strain encodes for three unusual substances: lysostaphin, surfactin and dispersin. These substances are biodegradable and already commercially available in a pure state for applications closely related to the one proposed here.


1. European Food Safety Authority (EFSA), 2010. Scientific opinion on the maintenance of the list of QPS microorganisms intentionally added to food or feed (2010 update). Panel on Biological Hazards. EFSA J 8(12):1944.
2. Morphogenesis of Bacillus Spore Surfaces, Venkata G. R. Chada, Erik A. Sanstad, Rong Wang, and Adam Driks, J Bacteriol. 2003 November; 185(21)
3. Role of the Spore Coat Layers in Bacillus subtilis Spore Resistance to Hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation, Paul J. Riesenman, Wayne L. Nicholson, Applied and Environmental Microbiology,Feb. 2000, p. 620–626

Biosafety group

Our institution (INSA Lyon) has, as every public institute, a general safety and health committee that deals, among others, with issues related to GMOs and that allowed their handling. All students have followed a 4-hour general health and safety course on how to handle chemical, biological and fire risks among others, completed by additional biosafety and lab training though the year by the professors, in relation to their course. Our institution does not have any specific biosafety rule but complies with all French biosafety regulations.

As far as the legal aspect is concerned, there is no specific legal framework for synthetic biology in France yet. Since our bacteria are Genetically Modified Organisms, their use is restricted by the legal framework about the use of GMOs, which is based on the precautionary principle. Even though synthetic biology doesn’t have a specific regulation framework yet, discussions are taking place about this issue at the French government and National Assembly levels to define a specific regulation. A first congress and public audition (Program) has occurred in May 2011.

After standard parts, why not a standard chassis ?

Beyond traditional physical containment (by restricting organisms to a physical space), disabling of the organisms in some way so as to ensure they cannot survive if accidentally or incidentally introduced into the environment. Such disabling leads to chemical containment (based on engineering "kill switches" or reliance on nutrients not found in the wild) or informational containment (by making genetic information incompatible between natural and synthetic species).
The Lyon-INSA team proposes to provide a “safety kit” to each team at the beginning of their experimental work. We think that if applied, this option would strongly diminish the contamination risks or gene dissemination into the nature.
This kit would contain a collection of “disabled chassis” for the most popular chassis (E. coli, B. subtilis…) that could be used to receive the DNA constructions.
As engineering these chassis is not easy, we suggest to create a new “ Best Safety Device” reward to motivate iGEM teams.


1 Contreras A., Molin S, Ramos JL. 1991. Conditional-Suicide Containment System for Bacteria Which Mineralize Aromatics. Applied and Environmental Microbiology.

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