Here you can find about how we stayed safe and prevented / managed risks associated with our project! It is important to note that our team had tremendous support from experts other than our supervisors who helped us manage our risks — Post-docs, lab technicians working at the university and Ph.D. students working throughout our lab
Our rEvolver team members were provided with lab coats, eye protection,, face masks, and gloves to avoid risks of contact with harmful chemicals. Face masks were originally mandetory due to a immunocompromised member of staff within the lab. At the end of each day, the lab space was also thoroughly cleaned with disinfectant to avoid unwanted microbial growth and further contamination.
Similarly, waste disposal was a large task for our team - we found ourselves generating a lot of waste through trial and error of our media optimisation experiments. Instead of disposing lab consumables, we were shown how to autoclave for safe use in the next lab session. Chemical waste disposal protocols were also thoroughly explained during our induction into the laboratories we worked in.
Online training courses are also compulsory for our wet-lab team during their degrees, so the core of our team was well rehearsed in the safety precautions present in the labs.
We worked in a biosafety level 1 laboratory whereby all our experiments utilised either E. coli (DH5-alpha or BL21) or V. natriegens (Vmax Express) which are all Risk Group 1 organisms. Further, cellular mutation systems have previously used UV radiation or harmful mutagens to drive diversity generation. We instead used an in vivo MutaT7 system, which utilises a T7 RNA polymerase fused to a base-editing enzyme and hence poses no risk to humans. Lastly, the new genetic parts that were constructed by the team were not inherently hazardous to human health.
The bioreactor design utilises a peristaltic pump to minimise human contact with the media and bacterial growth cultures, thus decreasing the chance for bacterial spread to skin or contamination of our laboratory equipment. Members of the bioreactor team were suitably trained in the iForge facility before conducting any work in the workshop.
We followed the Genetically Modified Organisms (Contained Use) Regulations of 2014, which applies to the use of GMOs in UK workplaces and research laboratories. The University of Sheffield Departmental Biological Safety Officer approved our project following these guidelines. Before beginning each protocol, we filled in a Control of Substances Hazardous to Health (COSHH) assessment which was then verified by our personal investigator, Dr. Hitchcock. All surveys carried out for our research were done in accordance with relevant laws and regulations here in the UK, as well as all institutional rules and guidance.
We initially planned to use mutagens but since we changed to the MutaT7 system, this is no longer required and is particularly safer for us humans! Highly flammable chemicals, such as ethanol, were kept away from open flames. SYBR Safe was chosen over ethidium bromide, which is highly carcinogenic to humans if in contact with the skin. PPE was also in place during every lab day to ensure safe use of each and every reactant we used during our project.
Upon initiation of laboratory projects, around Sept-Oct 2021, the entire wet-lab team underwent extensive safety and security training. Those that utilised the maker space were suitably trained in March 2022 and are also certified to train further members. The topics covered are as follows:
Since our project focuses on making in vivo directed evolution more accessible, it is therefore a possibility that deliberate misuse could lead to a bad outcome but it is extremely difficult to predict exactly what those bad outcomes may be.
Horizontal gene transfer is possible for our plasmids and DNA via bacterial methods (conjugation, transformation, or transduction via phage). However, our intention is to never use our system outside of a biosafety 1 laboratory and we discourage our users to perform our experiments in any kind of setting different to the biosafety 1 laboratory.