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 toxicity for example. For these reasons: the access of the laboratory was limited to those involved in the project and all work benches were cleaned every day and the whole lab cleaned every week.

CLEAN AREAS TO ENCOURAGE GOOD PRACTICES

Before starting experimental work, we have identified chemical/biological Hazards and Risks. We consider the fact of having a proper training in safety and security at the beginning of our experimental work prepared us to be more organised, responsible for our actions and respectful for those of others. To sum up: GOOD LABORATORY PRACTICES: PROTECT YOURSELF, PROTECT PEOPLE AROUND YOU, PROTECT THE ENVIRONMENT.

Global hazards and risks : Electricity and gas: we have followed all instructions from our institution concerning the lab electrical and gas systems. Emergency numbers are displayed near the phones. Personal protective equipement, including appropriate labcoats, gloves and safety glasses were worn for lab experiments. No one was allowed to work alone in the laboratory. In addition, the French system provide all young adults with a training in first-aid and safety. Moreover, several students, advisors and instructors have the life saving diploma and also have trained for firefighting.

Main chemical hazards and risks : Most of the reagents used are irritant, toxic and can be potential carcinogens (agarose, polyacrylamid, methanol, Ethidium bromide…)

To minimize the impact of their use, they are manipulated following the supplier’s instructions, wearing appropriate personnal safety equipement, i.e. gloves, safety glasses, labcoats and under extractor hood when necessary. All samples, tubes, vials are clearly identified/labelled to avoid inappropriate mix between two non compatible solvents. All reagents are eliminated in the appropriate waste recovered barrels.

We took specific safety measure for the use of Ethidium Bromide (EtBr). Ethidium bromide is known to act as a mutagen because it intercalates whithin the double strand DNA helix. Many biological processes can thus be affected such as transcription and replication. To avoid the dissemination of EtBr in the lab, it is stored and used in the same room where the electrophoresis gels are revealed. EtBr is NEVER incorporated into the electrophoresis gels, but used in staining bath instead to avoid the contamination of electrophoresis equipment. This room is locked by a key. The access to this room is forbidden to any person who does not carry a lab coat. Dedicated gloves are used for EtBr manipulation and must not leave the room. They are discarded in a specific trash barrel for genotoxines contaminated material to gather with stained gels. EtBr-contaminated material is further processed and decontaminated by an external service.

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.

Main biological hazards and risks: All Bacillus subtilis, Echerichia coli and Staphylococcus epidermidis strains we used have a biosafety level of 1, which means they are not known to cause disease and have minimal environmental hazards.

At this level the precautions concerning the lab-material are minimal: wearing gloves and face protection when needed. Any contaminated material is discarded in a trash can to be autoclaveded within 2 days. The decontamination procedure is similar to any other applied to frequently encountered virus or bacteria (ex: to wash hands with antibacterial soap, disinfect any surface in the laboratory that has been exposed). Our work benches are decontaminated with ethanol 70% after each manipulation. And contaminated media and materials are autoclaved at least every 2 days to avoid any release in environment.

All the students must have their vaccinations up to date. In case a student harbors an injury, it must be covered so it is not exposed to the bacteria. All rooms are equiped with with a first aid kit. In the building where the labwork was performed, no laboratory is manipulating pathogenic microorganisms, which limits the risks to students, and the recombination possibility between a pathogenic bacteria and the lab strains.

For better protection the following basic safety rules applied for all students during lab work:

• Be aware of the risks and hazards involved in any experiment
• Know where the fire extinguisher, safety shower and general electrical circuit breaker are located in each room
• No food or drink in the laboratory
• Wear a buttoned up lab coat
• Long hair need to be tied back
• Contact lenses are forbidden except if people wear a face protection or safety glasses
• Spillage need to be cleaned up immediately
• Lab benches are cleaned after each experiment
• Technical and safety files are available neat all the lab equipments
• No chemical touching, sniffing or tasting
• No mouth-pipeting
• No returning unused chemicals to their containers
• No hazardous material disposal down the drain

This specific localisation in a public research laboratory building allows us to benefit from researcher and student law safety (or under application in France) which are mandatory in each lab in France. For example, we have specific place for each kind of experimentation:

Rooms dedicated for:

• Bath
• Electrophoresis migration
• Incubators
• Freezer and refrigerator
• Autoclave

We use our personal autoclave in the lab to decontaminate and sterilize our materials.

A GMO may contain harmful parts and be dangerous for the environment. The potential danger of a GMO is also due to its potential interactions with the environment. When an iGEM project is conceived, the team has to think about the possible interactions of their parts with the environment and design tests to document the effects of their parts on the environment. Synthetic biology is a science which worries people, so we have to provide them the insurance of the complete safety of our experiments.

Our molecules : 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.

Our chassis

In our Biofilm Killer project, we aim to construct a bacterium able to destroy biofilms and to synthesize biocide molecules or a biosurfactant in order to develop a novel method to remove biofilms and clean surfaces in several industrial processes. So, we chose a strain that would have a minimum impact on the process itself, the quality of the product and biological treatment that could exists on site (waste water treatment plant).

Our genetic constructions have been transferred into a strain of Bacillus subtilis. This species are well known by researchers but also by many industries such as the food industry. Bacillus subtilis is also widely used in the field as a biocontrol agent against plant pathogens.

It has been granted Qualified Presumption of Safety status by the European Food Safety Authority (EFSA)



But Bacillus subtilis is also known to produce endospores that are resistant to different treatments (heat, UV irradiation…). A possible impact on human health cannot be excluded. Indeed, it has been shown that the ingestion of endospores of non-modified B. subtilis does not prevent their germination into the gastrointestinal tract (GIT) [4]. The bacteria are even capable of growth in the lower part of the GIT. But, in this particular case, the consequence seem to be beneficial on health. The presence of B. subtilis in the GIT seems to be linked to the development of the gut-associated lymphoid tissue (GALT). This is one of the reason B. subtilis can be used as a probiotic.

When the environment becomes unadapted to its growth, the bacterium induces the formation of an endospore in its intracellular compartment [2]. The core of the endospore contains the bacterial chromosome. The core is protected by a double layer membrane, called the forespore. This membrane is itself protected by the coat, composed by multilayered proteins. In B. subtilis, the Cot family proteins is widely involved in the formation of the coat. The resistance of the spore is dependant on the state of hydratation of the core. Spores can be dispersed in the environment and turn into germination processus leading to the contamination of new areas previously devoid of this micro-organism. In our case, we work with B. subtilis strain 168, which is the most widely used, and capable of sporulation, B. subtilis strain in research labs.

Sporulation can be hazardous to the environment and humans because spores are resistant to most cleaning procedure and thus can survive for a very long time, even in extreme conditions [3]. Spores can be dispersed in the environment and turn into germination processus leading to the contamination of new areas previously devoid of this micro-organism.

In our case, we work with B. subtilis strain 168, which is the most widely used, and capable of sporulation, B. subtilis strain in research labs.

The high spore resistance to adverse conditions imply that avoiding the induction of sporulation is one task that we need to consider in our project if we use a sporulant B. subtilis.

If the cells were released in the environment after the germination of spores, we estimate the chance of survival to be minimal, because strain 168 has been modified and habituated to laboratory conditions. Even so, as mentioned below, B. subtilis is not a pathogenic bacterium.

For all these reasons, and in the aim of an industrial application, we propose to introduce our genetic constructions in an NON-sporulant B. subtilis strain, such as QB1133.

The use of a non sporulant B. subtilis to host our genetic construction would not significantly impact neither the environment nor the human health. The odds of the bacteria surviving to the low pH of the stomach, and the very hard life conditions (anaerobia, pH, competition with the intestinal flora) are very low in the vegetative state.

In addition to the use of non sporulant mutant of B. subtilis, we could enhance safety 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"

Our institution (INSA Lyon) has a biosafety group. However, we have a general safety and health committee that deals, among others, with issues related to GMOs and that allowed their handling. All students follow 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 all along the year by the professors, in relation to their course. Our institution does not have any specific biosafety rule but complies to all the french biosafety regulations.

As far as the legal aspect is concerned, these 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 quite restrictive, based on the precautionary principle. Even though synthetic biology doesn’t yet 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, with not standard chassis ?

The INSA de Lyon team propose that a “safety kit” should be provided 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 should contain:

• a collection of chassis that could be used to receive the DNA constructions. The strains’ genome would be the result of the gene knockout method which is used to inactivate a specific gene. With the new genetic technologies that evolve each day, now it is even possible to purchase a commercial Gene Knockout System which can be designed to knock-out the gene of interest. For exemple, an applications of this genetical instrument is the construction of auxotrophic strains (or nutrient-deficient) which are mutant strains of bacteria that are unable to grow on minimal media. The proliferation of these organisms outside the laboratory is limited because the lab chemicals are not found in nature.
• a toxin/antitoxin system coupled with a lysing agent/cytotoxic compount/degradation agent which is provided on a separate backbone (it would let the choice to the participants whether or not they want to use it). If the strain containing this system is released, horizontal gene transfer between a non-natural bacteria and a natural one is prohibited. Thus, with the anti-toxin inside the chassis’ genome, and the toxin and cytotoxic compound in the plasmid DNA, the horizontal gene transfer will induce the death of the recipient.
• a strain suicide option represented by a gene-killer (also found on a backbone) which is activated in the absence/presence of a certain substrate. For example, Contreras et al. (1991) [1], proposed the construction of a confined strain capable of digesting polluted substance. In the absence of pollutant in the environment, a suicide gene is expressed and the cell is destructed.

We are aware that engineering these DNA sequences is not easy. This is why we suggest that a new section or a new reward should be proposed : “ Best Safety Device”. For teams it would be a real challenge and their investment for creating new safety devices would be rewarded.

In synthetic biology the safety and security measures are of primary importance. Every team should have a proper formation in safety and security issues. INSA de Lyon team suggests that one of the competition requirements should be a diploma validating a proper safety formation.