Safety and Security
A thorough and comprehensive safety concept is the foundation of every scientific project. As part of our iGEM experience, we sat together with experts from various fields and explored different aspects of safety and security, from personal protection against a dangerous toxin to potential ways to misuse our nanobody antidote.
General safety
As the biosafety level 2 lab of the Kolbe group used by our team is
situated at the Centre for Structural Systems Biology (CSSB), our whole
team participated in the general occupational safety and biosafety
training mandatory for conducting lab work at the facility. The training
includes rules and regulations regarding biosafety level 1 and level 2
labs.
Additionally, we received task-specific instructions for
working in the Kolbe group lab by our PI, Prof. Dr. Michael Kolbe. He
introduced us to the lab’s equipment, such as fume hoods, biosafety
cabinets, incubators, fridges and freezers, centrifuges, shakers and
photometers. He also showed us safety measures and their locations,
including emergency showers, eye showers, first aid kits and fire
extinguishers. Additionally, he showed us the assembly point in case of
emergencies. Only team members who completed both the general and
task-specific safety training received access to the lab in the form of a
transponder and were allowed to work in teams of two people minimum. All
lab procedures were conducted wearing the appropriate personal protective
equipment, such as lab coats and gloves for work with genetically modified
organisms (GMOs). Work with potentially dangerous chemical substances such
as trichloroacetic acid (TCA) for protein precipitation or acrylamide for
preparation of SDS-PAGE gels was conducted under the fume hood wearing
safety goggles, lab coats and gloves.
To handle liquid nitrogen for flash-freezing samples, we received additional special training for working with cryogenic gases, which included operating the filling station with a remote control for higher safety. All of our lab work is conducted in accordance with the Hazardous Substances Ordinance (GefStoffV), the Biological Agents Ordinance (BioStoffV) and the Genetic Engineering Safety Ordinance (GenTSV).
Working with GMOs
We chose two well-characterized standard E. coli strains (DH5α and BL21(DE3)) for our protein production and followed the guidelines for working with GMOs. We introduced a resistance against kanamycin to be able to select for positive colonies and isolate positive cells. Work with the bacteria was conducted using the appropriate lab and personal protective equipment, such as the biosafety cabinet, lab coats and gloves. All equipment in contact with GMOs is regularly disinfected.
To prevent potential spreading of antibiotic resistances, we collected all potentially contaminated waste and reusable equipment, which was sterilized through autoclavation prior to disposal or reuse.
Safety measurements regarding α-amanitin
To conduct analyses of the binding affinity of our designed nanobody, we planned on working with α-amanitin in the lab. As this toxin is highly dangerous to humans, with a LD50 of 0,1 mg/kg body weight, we needed to adhere to strict safety rules. Together with our PI Prof. Dr. Michael Kolbe and a specialist for work safety at the University of Hamburg, we created a risk assessment for handling α-amanitin in the lab. We used the knowledge gained in this process and information from safety data sheets to create operating instructions on handling, storage and disposal of the toxin. For more detailed information on the disposal of the toxin, we reached out to various groups in academia and industry working with amanitin or amanitin-containing samples (our human practice regarding safety ). We incorporated those inputs as well into our operating instructions. We did not end up working with α-amanitin due to time issues, but nevertheless deposited the created documents at the lab for future users.
Dual-use concerns of the project
In a workshop led by our PI and specialist on biosafety, Dr. Mirko Himmel, we discussed dual-use concerns and misuse of knowledge in science. He helped us to consider potential ways of misuse for our project and eliminate any obvious possibilities to cause harm. We plan on designing a nanobody specifically against α-amanitin using existing protein design and structure prediction tools and do not foresee any immediate risks of transferring the inhibitory effect onto other structures. As α-amanitin is a bicyclic peptide with hydroxylations, it is unlikely to resemble any human protein or peptide structures that could be bound accidentally. However, since we contributed to the accessibility of protein design and structure prediction tools by simplifying and annotating existing notebooks, we acknowledge that we might have contributed to potential misuse of these tools through more untrained users.
As one part of our wet lab approach, we prepared lipid nanoparticles (LNPs) to serve as a form of delivery for our nanobodies. We did not add any steps or procedures to the existing protocols used for the preparation, and did not load the LNPs with any cargo. Nevertheless, we acknowledge that by using existing, easy-to-use protocols and spreading them through our wiki, we might enable potential misuse by untrained users.
As our final product consisting of the nanobody against α-amanitin packaged into LNPs does not contain any living GMOs, our project does not pose any risks of accidental release into the environment.