Safety

Safety is fundamental in the development of genetically modified organisms (GMOs). To reduce the risks, it is important to experiment safely and responsibly by following laboratory safety rules.

Moreover, the handling of GMOs and micro-organisms in the context of laboratory research is highly regulated in France[1]. In this perspective, we were able to develop our experimental tasks thanks to the Laboratory of Quantitative and Computational Biology (LCQB), which benefits from a convention for the use of GMOs given by the Ministry of Higher Education and Research.

Biosafety and biosecurity

Although we have been authorized to handle GMOs, our main concern was to ensure that the modified organisms did not pose any risk to humans or the environment.

In our project, we introduced a gene from a prokaryotic organism into a eukaryotic photosynthetic organism. We chose to use a eukaryotic organism because they are non-pathogenic to humans, better controllable to avoid environmental and contamination risks.

The organisms are classified into risk groups [2] according to the danger they cause to humans and the environment (Table 1).

Risk group	Pathogenic for humans	Hazard to workers	Spreading	Effective prophylaxis or treatment	Safety equipment
1	No	Unlikely	Unlikely	Available	Open bench work
2	Can cause human disease	Likely	Unlukily	Available	Open bench work - BSC* for potential aerosols
3	Can cause severe human disease	Serious	possible	Usually available	BSC for aerosols - Other primary devices
4	Can cause deadly human disease	Very serious	Likely	Usually non available	Class III BSC - Positive pressure suit

Figure 1: Safety risk groups of microorganisms. *BSC = Biosafety cabinet (closed ventilated laboratory workspace).]

As we only used organisms belonging to risk group 1, there was no risk to the people working at the WetLab.

For cloning, we used the Escherichia coli DH5α strain. This strain is non-pathogenic and its impact on the environment is minimal. The potential risks associated with this bacterium are related to contact with the skin. We therefore handled this organism wearing gloves and a protective lab coat.

The UVM4 strain of Chlamydomonas reinhardtii is also non-pathogenic. Indeed, this microalga is a GRAS organism (Generally Recognized As Safe according to the FDA) and is therefore considered safe[3]. We still handled it while wearing gloves and a protective lab coat. However, we performed the algal cultures under a fume hood, not because of the risks involved but to protect our samples from contamination. Thanks to the laminar flow incorporated in the hood, we were able to protect our manipulations from any potential contaminants.

Safe project design

Our project was designed so that our modified C. reinhardtii is confined in a bioregenerative life support system (BLSS), so a closed system. However, there is a risk that our alga could end up outside this closed system. If this were the case, it would end up on the International Space Station (ISS) or on Mars, so there would be no risk of environmental contamination.

As for the risk to humans, a study has shown that the peptide we use is not toxic in mice and even provides protection against radiation. Although these results have been demonstrated in human cells they have yet to be confirmed in humans. Nevertheless, these results pave the way for potential protection[4].

Safe lab work

General laboratory safety rules

For a laboratory to function correctly, it is necessary to respect the common spaces (tidy up and leave clean the common work stations and living spaces), to respect the common work tools, and to know how to manage one's needs in work materials (small materials, consumables, culture media, sterilized materials).

The good practices in the laboratory are as follows :

Respect security instructions
Know the procedures to follow in case of fire or accident
Locate fire extinguishers and alarm buttons
Keep corridors, stairways and halls clear
Do not obstruct the closing of fire doors
Leave free access to the isolation valves (gas and water) and to the electrical cut-off devices
Close doors and windows when leaving your workplace. The last one to leave checks that the doors are closed
Wear the individual protection equipments (IPE)
Identify the risks – respect the labeling rules
Work in the right place (workstation adapted to specific risks)
Respect the procedures of the isolated worker

In case of professional risk (biological, chemical, radioactive), it is recommended to contact the professional risk prevention department by phone or by email. The Faculty of Science and Engineering also has an infirmary open every day.

Workspace

To work in the laboratory, we had to wear appropriate clothing (long trousers, closed shoes and tied up hair) and a lab coat. To avoid contamination of our samples, we changed our gloves regularly. Then, after each experiment, the samples were disposed of in the appropriate bins and we disinfected our bench to avoid any contamination.

We worked in different spaces with specific equipment. For each room, eating and drinking is forbidden. Certain protections may be necessary depending on the danger to which the experimenter is exposed in the room.

Laundry and preparation area: Ice machine, washing machine, MilliQ water, autoclaves, scales.

The hazards associated with this room are the presence of a pressure vessel (autoclave), the risk of burns, the electromagnetic field (microwave) and corrosive chemicals. In addition, this is a noisy area. In this room, wearing a lab coat is mandatory.

Machine and storage area: Liquid nitrogen tank, centrifuges, culture chamber, cold room, -80°C freezers, plate reader, qPCR machine, PFGE (Pulse-Field Gel Electrophoresis), pipetting robot.

This room presents a biological risk, a risk of asphyxiation, and a low temperature due to the presence of dry ice and liquid nitrogen. Protection of the body, face, eyesight, hands and feet is mandatory in this room. This space is air-conditioned, so the door must remain closed.

BET area : ADN/ARN electrophoresis, gel documentation system.

The hazards associated with this room are ethidium bromide (BET), UV radiation, and the electromagnetic field (microwave). Access to this room is restricted to laboratory staff and requires the wearing of a lab coat, gloves, and face protection when using the portable UV lamp.

The safety officers left us a summary of common sense practices to have in the BET room:

Always wear your lab coat and gloves when working in this room
Close the BET dropper bottle well and clean if necessary. Use 1 drop/100 mL TBE/TAE liquid, not more
Clean the UV gel box when finished with water or ethanol and put your gel in the BET dry waste bin
After staining your gel, put staining/destaining liquids into the BET liquid waste bin
After running a gel, cover and label the gel box you are using, noting how many times the same buffer has been reused
Use a support tray and tissue to remove your gel to take a photo. Do not leave TBE/BET drops on the floor. Clean up spills
Turn off the UV gel box when finished
Respect our co-workers by keeping this room clean

For the preparation of the electrophoresis gels, we used ethidium bromide. This fluorescent marker is a DNA intercalator and allows the visualization of DNA on the gel. This product is toxic and mutagenic, which is why we only used it in a dedicated room, wearing a protective lab coat and gloves to avoid carcinogenic risks. We received explanations from the laboratory's safety officers who showed us the specific bin in which to dispose of objects contaminated with the BET. Excess and non-recyclable solutions are then disposed of by a licensed waste disposal company. We also took care not to contaminate surfaces outside this room by changing our gloves before going into another room.

Evacuation

We have received safety instructions in case of evacuation. As soon as we hear the alarm signal :

Exit and follow the signs
Head to the gathering point or the secure waiting area (reserved for people with reduced mobility)
Do not go back
If there is smoke, get down
Before leaving, the person in charge makes sure that the windows and doors are closed

COVID-19 protocols

Due to the pandemic, we had to respect certain rules in order to work in a safe environment. The number of people in the lab being restricted, we limited our iGEM team members to 2 people to perform the manipulations. In order to respect this, we had set up a schedule to make sure that each day, 1 or 2 people would be present in order to make the experiments progress. We also had to sign authorisations for each member to work in the lab.

According to government guidelines, we applied the following barrier gestures:

Wearing a mask is mandatory (except for eating and drinking)
Wear one lab coat per person
Wash your hands very regularly
Cough or sneeze into your elbow
Use single-use tissues
Greeting without shaking hands
Keep physical distance (1.50 m)
Follow the directions of traffic
Maximum 1 person in the elevator cabin

As for the work outside the laboratory, we favored remote working sessions, or in the boxes at the university library, wearing masks. We also divided the tasks into small working groups to avoid being in a confined area with all the team members.

References

[1] O.G.M. en milieu confiné. Ministère de l’Enseignement supérieur, de la Recherche et de l’Innovation //www.enseignementsup-recherche.gouv.fr/pid28968/o.g.m.-en-milieu-confine.html.
[2] Use of Micro-organisms or Derivatives. https://www.esrf.fr/Infrastructure/Safety/Experiments/Biology_Experiments/use-of-micro-organisms-or-derivatives.
[3] Arias, C. A. D. et al. Semicontinuous system for the production of recombinant mCherry protein in Chlamydomonas reinhardtii. Biotechnol Prog 37, e3101 (2021).
[4] Gupta, P. et al. MDP: A Deinococcus Mn²⁺sup>-Decapeptide Complex Protects Mice from Ionizing Radiation. PLOS ONE 11, e0160575 (2016).

Team:Sorbonne U Paris/Savety

Lab Work