Team:INSA-UPS France/Safety

We can’t do anything we want in the lab. Regulations, rules of safety or restrictions have to be respected in order to keep the environment and the other safe..

INSA Safety Department

During the summer, we were received by the LISBP, a laboratory working on the biotechnology field. This structure has a safety department. Its goal is to ensure both the well-being of the employees and the respect of safety rules and risk prevention. All those topics are supervised by Nathalie Doubrovine, who instructed us, during a two times formation: an afternoon meeting, and an intranet formation through NEO software. We learned about fire risk, biological risk and chemical risk. At the end, we had a test to make sure that we understood everything.

Autoclave training

Our team has attended 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. The standard protocols of loading and unloading it were also demonstrated.

Legislation and French Labor Law

We are working in a public engineering school (INSA Toulouse), thus we have to respect the French national regulations about working conditions and 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 have 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.

Safety in the Lab

Basic Rules

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.

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

Three different cupboards were used to separate the 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 container for chemicals
  • Special container 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 biological safety cabinets (FASTER Ultrasafe). These BSC were cleaned with ethanol before and after each manipulation. A maintenance control is done each year. The last control was executed on December 12th 2016.

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 to use 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 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

Above, we have described the relevant security measures taken all through the 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 strains, which could be a threat to the public and the environment.

The E. coli and P. pastoris strains that we use are non-pathogenic and their genomes are very well characterized, thus we used them as our chassis. We also used Vibrio harveyi, a characterized bacteria, and opportunistic pathogen. This strain of Vibrio is characterized as a BSL1 organism, and can be used without restriction in the lab. Hence we chose to use Vibrio harveyi instead of Vibrio cholerae because of these considerations of pathogenicity and safety.

E. coli was used only for genetic manipulations, moreover the genes that were inserted (BioBricks) into this bacterium, did not increase its pathogenicity. P. pastoris and V. harveyi were the final active agents because of their ability to sense V. cholerae and the ability to produce AMP respectively. Therefore, the main threat is the dissemination of our bacteria in the external environment where gene transfers could occur between microorganisms. That’s why, to minimize both the risk of dissemination and 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), using a permeable membrane (XXX µm) in order to not let the DNA going inside drinkable water or a semi-permeable TPX bag.

We also planned that our antimicrobial peptide will kill both Vibrio cholerae and Vibrio harveyi and finally P. pastoris, destroying our system after use.

Chassis organisms used in our project

Bacteria Risk group Risk group source Disease risk for humans
Escherichia coli DH5 alpha 1 DSMZ No. These organisms do not cause diseases in healthy adult humans. (However, they might cause diseases in young children, elderly people, or people with immune system deficiencies.)
Escherichia coli K12 1 DSMZ
Escherichia coli MG1655 1 DSMZ
Vibrio harveyi JMH626 1 DSMZ
Pichia pastoris X33 1 DSMZ
Pichia pastoris SMD1168 1 DSMZ