General lab safety precautions
Lab safety instruction and education
Before we started the wet-lab work for our project, we received a detailed lab safety introduction from our PI, Dr. Nicole Gensch. Following this, we spent one week learning how to perform standard molecular biology protocols accurately and safely. Many of our team members did not have much experience in a research lab before this point, so it was important to get everyone on board with working in a sterile environment and avoiding contamination. To this end, good laboratory practice was taught, with proper waste management and disposal being an important focus. We chose two team members to be responsible for overseeing the correct implementation of the safety precautions, and as contact persons for all related questions.
Lab installations
Many of the biosafety, biosecurity and chemical containment installations in the lab were already in place when we started working there, as we were using the course labs in the shared wing of the institute. We used the centralized waste autoclave in order to deactivate our solid waste, and used a separate autoclave for our liquids and instruments. Our lab had three fume hoods, as well as secure cabinets for acids, alkali and flammables. For mammalian cell culture, we used the biosafety cabinets in the adjacent room. For S1 lab procedures such as cloning, work was performed on open benches, which were regularly cleaned and sterilized with 70% ethanol.
Personal protective equipment (PPE)
Before we started working in the lab, we made sure that all team members had access to their own lab coat. Hangers for the lab coats were provided in the corridor leading to the lab rooms. Furthermore, all team members were instructed to wear closed shoes and long pants during active lab work, and single-use gloves were provided in three different sizes. Additionally, a UV face shield was provided for UV protection when working with the gel transilluminator.
Work safety procedures
Bacterial strains and culture
For most of our lab work, we used the standard Escherichia coli laboratory strains DH5ɑ, DH10β and BL21(DE3). These strains are standard for molecular biological operations, such as cloning and T7 expression, and do not pose any undue risks to the lab workers. For more specific applications, we used E. coli strains BTH101 from Euromedex and bMS.346, which we acquired from Addgene. When working with any of these strains, we followed sterile working conditions, which meant that single-use gloves were worn, and all utensils for working with the cells were either single-use, or could be sterilized using a flame immediately before and after use.
Mammalian cell culture
For our research work we used U2OS and HeLa cells. We made sure to get safety certificates for all cell lines used during our research. Our mammalian cells were handled with single-use gloves and at all times, lab coats were worn during all mammalian cell culture activities, and the culture flasks and reagents were only opened in our biosafety cabinets. After every activity in the biosafety cabinets, we disinfected the working area thoroughly using an ethanol solution, and after the work was completed, we further disinfected by 2 hours of UV irradiation throughout the entire biosafety cabinet.
Repebodies
Since Repebodies are still relatively obscure in research use, we had to consider the safety of using these proteins, especially since they contain a domain from the pathogenic protein Internalin B (InlB), found in Listeria monocytogenes. This domain, however, does not show any function other than being a structural element itself, and is therefore most likely harmless.
Reagent and chemical exposure
For our project, we worked with several chemicals that pose potential health risks for their users, such as SDS powder, non-polymerized acrylamide and Coomassie. Furthermore, strong acids and bases were used, such as hydrochloric acid and sodium hydroxide. We worked exclusively in the fume hoods with all chemicals that can irritate the mucous membranes or be toxic when inhaled.
Waste Management and Disposal
As a central part of our biological and chemical containment procedures, we sorted our waste, and disposed of it according to its origin and hazard level. For most biological S1 waste, this meant collection and autoclaving in the central autoclave. We collected our agarose gels separately from our other biological waste, as they still contained the intercalating fluorescent dye used to stain DNA, that is a potential carcinogen. There were separate containers for broken glass waste (contaminated) and used sharp objects, such as scalpels and injection needles. In our fume hoods, we had a neutral-pH chemical waste container.
Downstream risks
As part of our ongoing biosafety and biosecurity efforts, we have considered which hazards may be posed by our research, and by the completed product of our project.
Biosafety
As with all biological research products, there are concerns about unintentional release into the environment, or accidental infection of laboratory staff with our product. As our project does not use viruses or bacteria other than E. coli, and does not give our bacterial cultures a fitness advantage over wild-type E. coli, we do not expect our work to pose a safety threat, especially if correct sterile working practice is used while working with our cells. Most likely, any cells which escape containment would either be outcompeted, because they contain mutations or gene knock-outs that would put them at a disadvantage, or they would lose their genetic modifications due to a lack of selection pressure from antibiotics.
Nevertheless, it is worth considering what further safeguards may be implemented, in order to further reduce possible safety threats. To this end, it was suggested to us by PD Dr. Boldt, our resident bioethics expert, to install a temperature-sensitive kill switch, which would deactivate any cells that get heated to 37˚C, such as inside a human gastrointestinal tract, effectively preventing opportunistic infection by the lab strains. Some of our parts already exhibit a similar behavior, pSC101 is a temperature-sensitive origin of replication that drastically reduces the plasmid copy number as the temperature approaches 37˚C. These temperature sensitive kill switches could then be periodically inspected, by growing the cells on antibiotic-free media at 37˚C, and observing the colony density.
Biosecurity
One issue which may arise from our combination of targeted protein degradation, as well as our Repebody generation system, is that malicious actors may design targeting chimeras, which are able to degrade critical proteins in human cells, and thereby create a harmful product. This hazard is at odds with our commitment to open science, since it results from the deregulated access actors may have to our products. A classical approach to minimizing such risks would involve either governmental oversight over the production of Repebodies using our method, or an industrial consortium regulating its use.
At the time of writing, our project does not have the parts necessary to become a biosecurity threat, and its development to such a level would certainly require significant resource and time investment. We wish to maintain our commitment to open science, but acknowledge the potential risks our project may pose when completed, and urge researchers building on our work to keep the mitigation of biosecurity risks in the forefront of their efforts.
One option to minimize the biosecurity risk described above, proposed to us in a meeting with PD Dr. Boldt, would be to restrict access only to humanized, or similar non-immunogenic CONCAVE kits. Since in the final product the retrons and Repebody would ideally be fully integrated into the bacterial genome, it would likely be more difficult to replace an immunogenic Repebody and the associated retron donors, than to use a different technology for malicious purposes, making these immunogenic CONCAVE kits non-problematic from a dual-use perspective. The trade-off for this approach would be that human therapeutic Repebody development would be more difficult, since the humanized CONCAVE kits would be contained and have public access restricted.
