Team:Toulouse/Safety

"Safety is not just a slogan, it's a way of life"

iGEM Safety

We filled the iGEM safety form with the help of Nathalie Doubrovine, safety officer at the LISBP.

Chassis organisms

New and/or modified coding region

Team safety

Safety in INSA Toulouse

INSA Toulouse is a public school for engineers. The biosafety guidelines are not specific to our institution; the French National regulations for working conditions and for manipulating genetically modified organisms are applied.
The regulation about the workers' prevention against risks resulting from their exposure to pathogenic biological agents (Decree No. 94-352 of 4 May 1994) includes microorganisms, cell cultures and 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.)
The Decree of the 16th July 2007 describes the technical preventive measuresto set up in research laboratories (including containment), education, analysis, anatomy and surgical pathology, autopsy rooms, and industrial and agricultural facilities where workers are likely to be exposed to biological pathogens.
The rules of health, safety, and preventive medicine applied in public services in France (and thus in all public facilities working in scientific and technological domains) are set out in the Decree No. 82-453. This decree refers to the Labour Code, Public Health Code and Environmental Code.
The Decree No. 2011-1177 is related to the use of genetically modified organisms.

There is no one in charge of the biological safety at the INSA Toulouse. However there is an organization which includes one prevention advisor and several prevention assistants in every Laboratory. As far as the LISBP (our structure) is concerned, Nathalie Doubrovine is the prevention assistant.
We have already discussed our project with her. She has given us the safety training and advices about how to respond to an imminent danger.
She raised the problem of GMO and the related policy. She advised us of the special procedure for the destruction of GMOs: we cannot wash this kind of organism down the drain.
We were also informed about the potential chemical and microbiological risks.

The laboratory safety training requirements of the LISBP are detailed into the Rules of Procedures of the LISBP.
The legislation requires employers to inform "all new employees" of the risks that they may encounter as well as ways to protect themselves from those risks. Therefore any newcomer must follow an appropriate training and pass a practical test about safety to ensure its own protection as well as protecting his/her colleagues.
Every person that enters in the LISBP has to make this training whatever its status (researcher, PhD student, trainee, etc.).
The training is divided into two parts. The first one is a training concerning general risk prevention in research laboratories. This one is made individually with the NEO software and with the explanations of the prevention assistant. The second one is a training about the techniques used during the occupation.

We also had a training to learn how to sort the different sorts of biological waste and how they are destroyed. We also learned that we cannot use autoclave on our own because a specific training is needed. We did not do this specific training and hence did not touch the autoclave. Only our supervisors could start these autoclaves...

Safety in the lab

"If you don't think it's safe, it probably isn't"

We have organized our workspace. We have a relaxing room where no biological material should enter. In this room, we can eat and drink (a fridge, a kettle and a coffeemaker are available) but it is also the room where we have our meetings and where we can work on our computers. On the contrary, in the lab, we have to wear protective equipment and respect basic rules.

Personal protective equipment

As soon as we manipulate in the lab, we have to wear the following personal protective equipment:

- A conventional lab coat, closed with long sleeves
- Closed shoes
- Gloves
- Glasses if needed (UV exposure, hot water or chemicals manipulation.)

Basic rules in a lab

We have to apply the basic safety principles into a laboratory room:
- 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 with the use of an electric burner.
- In some cases (UV light, projection risk), it is compulsory to wear protection glasses.

Different apparatus are used to work a sterile area.

Waste

Different trash containers are available in the lab:
- One for biological waste (yellow). This waste will be autoclaved before being thrown out.


- One for common waste (green or orange).


- Special waste for chemicals.

Devices and Material

We use a lot of different devices, and each one involves a particular risk. Here we described how we use this material safely to reduce risks:

Chemical storage

We have three cupboards dedicated to the different kind of chemical products we use:
- Flammable


- Acids


- Bases


Those cupboards are key-closed.

Ethidium Bromide

We have a dark room dedicated to the use of EtBr and UV. This room is key-closed and everyone entering into the room must wear gloves, glasses and lab coat. Everything which is in direct contact with something in this room has to stay here.




Two specific trash cans are dedicated to the gloves or paper and the agarose gels contaminated.

Biological safety cabinet

We use a Biological safety cabinet (FASTER – Ultrasafe) to manipulate into a sterile area and thus avoid external contamination by unwanted microorganisms. We clean the bench of the BSC with ethanol before and after each manipulation. We also clean the BSC completely every two weeks. Because we work with fungi, we have to be very careful with the cleanliness of the BSC.

Electric burner

We also use electric burners to manipulate into a sterile area. The burning and fire risks are high but weaker than with a Bunsen Burner. We are very cautious when we use electric burners, and we use in preference BSC if it is possible. We do not have to wear gloves and we have to check that the electric burner is turned off after the manipulation.

Chemical hood

We use chemical hood in case we have to manipulate dangerous volatile chemical compound.

Water-bathes

The water-bathes are used extensively (transformation, digestion, etc.). However, they can be dangerous because of the exposition of boiling or hot water. We use special gloves for protecting us from heat, steam and projections. We verify that we have turned off the water-bathes at the end of the day.

Safety of our project

"Precaution is better than cure"

Several risks exist when working with microorganisms and manipulating genes. We can identify different classes of risk of our project now:
- risks to the safety and health of team members, or other people working in the lab,
- risks to the safety and health of the general public,
- risks to the environment,
- risks to security through malicious use by individuals, groups, or countries.

There are also risks in the Future, linked to our project’s growth (new knowledge and methods development).

Risks to the safety and health of team members, or other people working in the lab

We work with the B. subtilis and E. coli chassis. These organisms are non-pathogenic. Moreover the used parts are also non-pathogenic (the bio-fungicides produced are harmless for humans).
On one hand, there is a major biological risk because of the manipulation of DNA and RNA of bacterial organisms.
On the other hand the main risk for humans is chemical: we use Ethidium Bromide, hydrochloric acid, sodium hydroxide, solutions from a Miniprep kit, ethanol and bleach.

However, we have a black room dedicated to the manipulation of Ethidium Bromide. Everything that touches something in this room cannot get out of the room. The waste has a special treatment.
For the other chemical solutions, we always use gloves and glasses, and if necessary a hood.
Furthermore, there are other risks such as UVs when we analyze an agarose gel but also water-bath and electrical/fire risks.
For protecting us from the UVs, we use adapted glasses.
At the end of every day, the last persons to leave check that everything is ok in the lab (no water-bath stayed on, everything disconnected).

Risks to the safety and health of the general public

If biological materials escape from our lab, the risks regarding safety and health of the general public is low because the manipulated parts are harmless. The only risk is the contact with DNA or RNA from a bacterial organism.
Moreover, we have to be cautious with the use of antibiotics and we must not wash them down the drain.

Risks to the environment

If biological materials escape from our lab, GMOs can spread in the environment and gene transfer can occur. Moreover, the aim of our bacterial system (called SubtiTree) is to kill a fungus (Ceratocyctis platani). The risk to the environment is high because our system could perturb ecological niche and unbalance the environment of some plants. All our waste is autoclaved to minimize the risk for the environment.
We use three fungicides.
The first one, D4E1, is a synthetic peptide.
The second is EcAMP-1 (BBa_K1162001), an AntiMicrobial Peptide from banyard grass added to the Registry last year by the team Utah State.
The last one is GAFP-1, the Gastrodia AntiFungal Protein.
These fungicides are not authorized yet by the European Union because they are not commercialized.

Risks to security through malicious mis-use by individuals, groups, or countries

If someone steals the biological material it could be dangerous for the environment because our bacterium will produce three bio-fungicides. This could lead to the disturbing of some ecosystems.
However, the fungicides are natural and one of them is used in agriculture. With these considerations, we can assume that the risk is not very high for the chosen parts.

How to reduce these risks?

We take care of our waste and our biological material.
We have chosen only non-pathogenic species to work on: E. coli and B. subtilis.
We use non-pathogenic fungus to test our SubtiTree system: Aspergillus brasiliensis, Chaetomium globosum, Trichoderma reesei and Aspergillus nidulans.
Indeed the targeted fungus, Ceratocystis platani, is pathogenic. Therefore we avoid working with it.

Risks in the Future

The system designed could be applied to many fungi. The fungicides used in our project are biological and specifically kill the fungi, and no other eukaryotic cells.
However, a malicious person could replace these fungicides by others which are more dangerous. Indeed our problem is that there is no regulation of the production of fungicides.

How to reduce these Future risks?

We have thought about several ways to minimize risks:
- we can build an auxotrophic strain of B. subtilis. The auxotrophy will be for proline, which is readily present in the plant's sap.
- we can build a non-sporulating strain ofB. subtilis. Sporulation is a bacterial mechanism used to survive through winter. Thus, our bacterial system sould not stay in the tree after the first year cycle.
- we can build a toxin/antitoxin system to avoid gene transfer between our optimized bacterium and any other wild-type bacteria.