Difference between revisions of "Team:Toulouse France/Safety"

Abstract: In the lab, we have to respect numerous safety regulations for the security and health of others and the environment.

Presence of safety department in INSA

During summer, our team worked in the engineering school INSA, which has a safety department. Their goal is to ensure the well-being of the employees, regarding safety rules and risk prevention. In our laboratory, the LISBP, safety is supervised by Nathalie Doubrovine who was the one instructing us regarding safety procedures. Thanks to her, we have completed a formation (with the NEO software) to learn the different risks related to our work in the laboratory and how to react in case of danger. The formation contained four parts: prevention, fire risk, biological risk and chemical risk. At the end, we had a test to make sure that we comprehended everything.

--> Autoclave training: Our team has taken an autoclave training, which showed us the explosive and implosive dangers of the dispositive and the security measures to take in order to protect oneself. A lab coat, heat resistant gloves and glasses were required for the manipulation of the autoclave.

--> Legislation and French Labor Law: We work in a public school for engineers (INSA Toulouse), thus we have to respect the French national regulations about working conditions and the manipulation of genetically modified organisms. As we work with microorganisms and cell cultures, we are concerned by the regulation on workers’ protection against risks resulting from their exposure to pathogenic biological agents (Decree No. 94-352 of 4 May 1994). It also includes human endoparasites which may cause infections, allergies or toxicity. This Decree is the French transposition of the Directive 90/679 / EEC and is also transcribed in the Labour Code (Articles L4421-1 R4421-1 to R4427-5). This Decree of the 16th July 2007 describes the technical preventive measures that are to be followed in research laboratories, where workers are likely to be exposed to biological pathogens. We have to obey to the rules of health, safety, and preventive medicine applied in public services in France (Decree No. 82-453). This decree refers to the Labour Code, Public Health Code and Environmental Code.

Basic rules in a lab

We have to apply the basic safety principles in all laboratory rooms:
- It is forbidden to smoke in all rooms.
- It is forbidden to drink and eat in the laboratory rooms.
- It is compulsory to wear a closed lab coat in cotton.
- It is compulsory to wear closed shoes.
- Long hair must be tied back.
- Oral pipetting of any substance is prohibited in any laboratory.

There are also others precautions when working with biological organisms:
- We need to wash our hands regularly.
- It is compulsory to wear gloves except for the use of an electric burner.
- In some cases (UV light, projection risk), it is compulsory to wear protection glasses.

Safety in the Lab

- Equipments: As soon as we manipulate in the lab, we have to wear the following personal protective equipments (a conventional lab coat, closed with long sleeves, closed shoes, gloves, glasses if needed).

- Storage: 3 cup-boards for different kinds of chemical products: flammable, acids, bases.

- Waste: Different trash containers are available in the lab:
- One for biological waste. This waste will be autoclaved before being thrown out.
- One for common waste.
- Special waste for chemicals.
- Special waste for waste in contact with ethidium bromide.

- Biological safety cabinet: To work into a sterile area and thus avoid external contamination by unwanted microorganisms, we have used a biological safety cabinet (FASTER – Ultrasafe). This BSC was cleaned with ethanol before and after each manipulation. A control of maintenance is done each year. The last control was executed on October 25th 2015.

- Water-bathes: We often used the water-bathes for transformation or digestion, etc. They can be dangerous because of the exposition to hot or even boiling water. To protect us, wearing a lab coat and special gloves was indispensable. The same equipment is necessary for the microwave.

- Ethidium Bromide: For some manipulations (to reveal gel electrophoresis), we have used ethidium bromide and UV light. Thus, a dark room is dedicated for that. This room is key-closed and the wearing of a lab coat, gloves and glasses is mandatory. Everything in direct contact with something in this room has to stay there. Wastes are treated specifically. A specific trash can is dedicated to gloves or paper and another is dedicated to the agarose gels that were contaminated.

- Chemical hood: We used the chemical hood when we had to manipulate dangerous and volatile chemicals (for example: the usage of alcohol 96°)

Safety for our project

We have described above the relevant security measures taken all through summer to minimize the risk of incidents in the lab. However, when working in the field of synthetic biology, one of the main concerns is the dissemination of our engineered strain, which could be a threat to the public and the environment.

We have used E. coli and B. subtilis chassis, since their genomes are very well characterized and, of course, since these bacteria are non-pathogenic. E. coli was used for genetic manipulations, moreover the genes that were inserted (BioBricks) into this bacterium, did not increase its pathogenicity. B. subtilis was the final therapeutic agent because of its natural presence in the cave’s microbiote. Therefore, the main threat is the dissemination of our bacteria in the external environment where gene transfer could occur between microorganisms.

This is the reason why, to prevent any risk of dissemination, all our waste was autoclaved before being appropriately disposed. Moreover, the French regulation does not allow any genetically modified microorganisms to be taken outside the lab. We are therefore aware that it won’t be possible to test our genetically modified bacteria in real conditions. In fact, it is unthinkable to release our bacteria in the environment without being confident enough that there won’t be any disturbances of the ecosystems of the Lascaux caves and the surrounding environment.

That’s why, to minimize both the risk of dissemination and of gene transfer, several solutions were planned. First of all, we have designed a physical device that would confine our engineered bacteria to the area to be treated (see the device part).

Apart from this physical containment, we also planned of adding two toxin/anti-toxin systems that would considerably decrease the probability of gene transfer (see project section). We also thought of including a biological clock that would limit the lifespan of our therapeutic bacterium. Moreover, we planned to use a spo- B. subtilis strain to prevent the bacterium to form spores that would allow it to resist to any competitors and to poor nutriment conditions.

Chassis organisms