Safety Training
The Wetlab team participated in a mandatory biosafety training course before entering the laboratory. This included instructions in disinfection, sterilization, emergency procedures, waste disposal, and handling of chemicals among others. The course was taught by the health and safety representative from the Research Unit of Molecular Microbiology at the University of Southern Denmark: Eva Lillebæk. The safety training applied to the laboratory at the Research Unit of Molecular Microbiology and the Department of Green Technology.
At the start of September, the Wetlab team renewed their safety training at the Department of Green Technology. This course consisted of a series of videos and reading material followed by a test. This test was passed by all members of the Wetlab team.
Laboratory Facilities
Throughout our project, we have worked in biosafety level 1 (BSL-1) laboratories. Because the project is a collaboration between different departments at the university, our laboratories were far apart. Therefore, we received training on how to move gene modified organisms (GMO) and chemicals responsibly between the laboratories. The transport of GMOs was done in containers labeled “GMO Class I transport”. After safety training, our team members gained key card access to the laboratories.
We did not have personal access to our biosafety level 2 (BSL-2) laboratory. However, due to our departments fluorescence microscope and French press being in their BSL-2 laboratory, we had access under the supervision of one of our supervisors/PIs.
Our work was carried out on the laboratory workbench, under chemical fume hoods, or in biosafety cabinets when necessary.
Antibiotic Resistance
To be able to select the correct assembly and transformation of the plasmid, we used kanamycin-, ampicillin-, and chloramphenicol resistance genes as selection markers. Using antibiotics in the laboratory carried the risk of spreading antibiotic resistance to wild-type bacteria outside of the laboratory, for instance, via horizontal gene transfer. The risk of such an occurrence is small since we were following the guidelines for working with GMOs.
The guidelines included proper disposal of live GMOs in autoclavable plastic bottles and disposal of contaminated single-use tools into properly marked GMO waste bags, which must be autoclaved. They also included guidelines for cleaning of multiple-use tools. For example, bacterial waste in flasks should be emptied into bacterial waste jugs, then rinsed with 70% ethanol into the same container, collected, and lastly cleaned in tubs with iodophor.
Safety Considerations
The parts that we decided to work with were dehalogenase enzymes originally found in Delftia acidovorans that can break down PFAS. As of right now, there is no evidence that these dehalogenase enzymes can be harmful to humans, animals, or the environment.
When we decided which organism to work with for our project, we briefly considered using a Pseudomonas strain, because they are naturally highly tolerant to fluoride1. The reason for initially wanting a microorganism with a high fluoride tolerance was (1) to ensure the bacteria survived the selection process for the most efficient enzymes and (2) to keep the opportunity of making a PFOA-filter with live bacteria open because some of the enzymes we worked with (DeHa 2 and 4) release fluoride when they break down perfluorooctanoic acid (PFOA).
Had we decided to use a Pseudomonas strain, it would not have been necessary to make them more fluoride tolerant. Some Pseudomonas strains, like P. aeruginosa or P. maltophilia are opportunistic pathogens2. Working with these requires a BSL-2 laboratory, which would increase the health risks for our team and require more advanced training. Therefore, we decided to use different E. coli strains. We worked with a number of different E. coli strains due to their different genomic features. The strains we worked with were E. coli Rosetta, E. coli Top10, E. coli BL21, NEB T7 Express Competent E. coli, NEB 5-alpha competent E. coli and NEB Stable Competent E. coli.
With our supervisors, we decided that E. coli would be able to express the genes we were interested in, and we would do additional experiments to make E. coli more tolerant to fluoride. The E. coli strains that we used are not pathogenic and therefore less risky to work with.
In our project, we worked with the chemicals PFOA and sodium fluoride (NaF) which could be harmful if not handled properly. We researched the chemicals extensively using a website called Kemibrug3 and therefore knew how to handle them properly. Both chemicals are toxic and were always handled under a fume hood, when in powder form or steaming solutions. In both cases, chemical gloves were worn. Furthermore, PFOA is corrosive and can cause serious health hazards.
Despite the health concerns, working with these chemicals was necessary for us to reach our goals for this project.
Everyday safety in the laboratory
For everyday laboratory work, we did the following:
- We disinfected our workbenches with 70% ethanol when work was done for the day to avoid contamination of the workspace.
- We got rid of GMO waste in the appropriate GMO waste baskets so they could be autoclaved.
- We made sure to get rid of chemicals in the appropriate waste containers according to waste groups, so they were handled safely.
- We used appropriate safety equipment when handling bacteria and chemicals. This included always wearing a lab coat and gloves when handling bacteria. This was for our safety, so we did not spill on ourselves or contaminate our samples.
- When handling NaF and PFOA, we used chemical grade gloves and safety goggles. Working with NaF and PFOA in pure powder form, or casting plates with NaF or PFOA in a hot solution always happened under fume hoods.
- If any doubt did arise during our laboratory work, we made it a habit to always “ask twice, do it once”. This both lessened the risk of wasting materials on a failed experiment and ensured both the safety of ourselves and our colleagues.
- Our GMOs were stored in an access-restricted -70℃ freezer.
- There were no fluorescence microscopes in BSL-1. Therefore, we had to outsource the fluorescence measurement of cells to BSL-2. Because we did not have access to BSL-2, one of our PIs, Lin, generously offered to help us do this task.
- Likewise, the French press, which was being used to break down our cells, also took place in BSL-2 under supervision. The cellular debris was then transported responsibly back to BSL-1, where we could perform protein purification.
- The FACS machine was located at another unit of the university, so we needed to transport the samples there.
Personal data safety
Looking at the General Data Protection Regulation (GDPR) laws, we ensured that we handled personal data safely. We did not collect personal data such as personal IDs, health insurance cards, and so on. Whenever we consulted people, we informed them that their answers would be used in our project.
When filming people, we obtained their written consent via email, and we made sure that personal pictures without consent were excluded from the Wiki. While creating videos, we also asked our respondents if they wanted to preview the video before we added it to our Wiki so they could approve. A few of our respondents accepted this.
