Overview
Since we chose our project PFAway, which focuses on the degradation of PFAS, it was essential to prioritize safety when handling these persistent pollutants. Our approach to safety was twofold:
1. Protecting people: ensuring that all team members could work in a safe environment.
2. Protecting the environment: through proper waste management to prevent the release of PFAS and GMOs.
We worked under the supervision of prevention and safety officers at Toulouse Biotechnology Institute (TBI) and we received a safety training to ensure that all experiments were conducted and materials were handled in accordance with both national and European regulations.
Safety meetings and training
All these actions helped us manage the risks in the laboratory.
Laboratory Safety
French legislation
As we work in a French laboratory, we must proceed in compliance with the French legislation, in particular the Orders of July 16, 2007 [1] and December 27, 2017 [2] fixing the technical measures of prevention, in particular of containment for places such as laboratories of research, analyses and teaching, where people are likely to be in contact with biological pathogenic agents (NOR: MTST0756429A and MTRT1633568A). In addition, the decree of March 7, 2008 on the prevention of biogenic risks (Articles R4421-1 to R4427-5) [3], present in the French labor code has to be respected. More specifically for GMOs, the order of March 28, 2012 imposed on us to make a file for contained use of GMOs according to the article R. 532-6, R. 532-14 and R. 532-26 of the environmental code [4].
Lab material and equipment
We conducted all experiments in a Biosafety Level 1 (BSL-1) laboratory, equipped with open benches, biosafety cabinet and chemical fume hoods to ensure safe handling of biological and chemical materials.
We used the autoclave to sterilize laboratory materials, such as bottles and pipette tip boxes, as well as to decontaminate biological waste before disposal. Protective equipment, such as heat-resistant gloves, lab coats, and safety gloves were worn during handling.
Biological Safety
Choice of our chassis
Escherichia coli, Pseudomonas putida, and Labrys Portucalensis are classified as BSL-1 organisms, meaning that they are non-pathogenic and safe to handle in a Microbiological Laboratory level 1 environment.
However, Pseudomonas putida, although classified as a BSL-1 organism, is an opportunistic pathogen and some strains have been reported to infect humans with compromised immunity (nosocomial cases). Special care was then taken when handling P. putida.
For all microorganisms, we followed BSL-1 laboratory procedures: wash hands after handling biological materials, decontaminate work surfaces, autoclaving of biological waste and no release into the environment. We were aware that an error in manipulating the bacteria or when testing them may cause accidental exposure.
Origin of our biological parts
| Gene | Organism of origin |
|---|---|
| TP | Bacteriophage PRD1 from Escherichia coli |
| ODNAP | Bacteriophage PRD1 from Escherichia coli |
| DSB | Bacteriophage PRD1 from Escherichia coli |
| SSB | Bacteriophage PRD1 from Escherichia coli |
| Dehalogenase | Delftia acidovorans |
| fluC | Pseudomonas putida |
| Fluoride-specific sensor | Organism of origin |
|---|---|
| crcB fluoride riboswitch | Bacillus cereus |
| crcB fluoride riboswitch | Thermotoga petrophila |
All the parts used were safe (no toxin) and were either provided by IDT as synthetic DNA or by academic researchers from our partner laboratory TBI, or were directly amplified from genomic DNA.
Chemical Safety
In our project, we worked with PFAS (PFOS, PFOA, PFPrA, TFA) and other fluorinated molecules (NaF, 2-fluoropropionic acid), known for their high chemical stability and toxicity. Failure to handle these chemicals according to the required safety precautions may cause accidental exposure.
To ensure the safety of all the team members and prevent any risk of contamination, we have implemented strict handling measures:
- Systematic use of nitrile gloves, lab coats, and safety glasses during all manipulations.
- All experiments were carried out under a fume hood to avoid inhalation or accidental dispersion for the preparation of stock and intermediate solutions, and microplates containing bacteria.
- PFAS solutions were stored in clearly labeled, air-tight containers under a fume hood.
- Microplates transportation out of the fume hood was carried out in a hermetic box.
- Dedicated pipettes were stored and used exclusively under the fume hood to prevent cross-contamination.
In all cases, when handled outside the fume hood, the quantities of PFAS transported in Erlenmeyer, flasks or microplates were under toxicity thresholds for inhalation and ingestion.
PFAS should harm the environment if the contaminated waste is not handled properly and the chemical is accidentally released. Therefore, a proper waste management system was implemented (Figure 1).
GMOs take precedence over chemical waste. Therefore, any waste that has been in contact with both fluorinated compounds and genetically modified organisms is treated as GMO waste and disposed of in the infectious waste in.
Other experimentation-associated risks
We used Ethidium bromide (carcinogen, mutagen) to stain DNA for gel electrophoresis, and ethanol (flammable) to purify DNA and for miniprep.
We also used antibiotics (ampicillin, kanamycin, streptomycin) to select our modified strains and m-Toluic acid as a transcriptional inducer.
Due to the chemical hazards involved, we followed appropriate safety measures, including wearing lab coats, and when necessary, gloves and safety goggles. Failure to comply with safety rules when manipulating these molecules would cause accidental exposure.