Working with a virus, especially a genetically engineered one, has been a concern from the start, both to ourselves and people we have talked to about our project. For this reason, safety and security has been on our minds since the beginning. There were several steps that were taken in order to minimize any risk associated with our diagnostic kit.
Firstly, M13 was chosen, among other reasons, due to its safety profile. It is a BSL-1 organism, infecting only F+ strains of E.coli (also a BSL-1 organism), unable to cause human disease. Also, among phages, due to its lysogenic life cycle and error-free replication, it could even be used in vivo without harm to mammalian cells. [1]
Secondly, M13KE (New England Biolabs) was chosen for the phage's genome. This is a cloning vector not containing any antibiotic resistance genes, instead relying on blue-white screening for transformed colony selection. We consciously decided not to introduce any, either, to minimize potential spread of antibiotic resistance in a country already suffering from drug-resistant pathogens.
Another important consideration is the specific modification we went for. The pVIII protein does not take part in the phage's infectivity, nor is it toxic in any way. Our modifications on this structural protein would probably make the engineered phages less robust than wild-type phages, leading to them being outcompeted in the long run in the event of a breach of containment.
Before starting our experiments we were trained by our Instructor, Elena Pappa, member of the Special Technical Laboratory Staff of the genetics and biotechnology department of the University of Athens. She taught us how to use the laboratory equipment safely, conduct experiments in sterile conditions and protect ourselves, giving emphasis on the most dangerous potential hazards, such as UV light and toxic chemicals. We were also informed about the guidelines we have to follow in case of emergency, like fire, gas leak or someone being wounded.
Apart from that, we also attended the Biosafety Seminar - Training delivered by Dr Thanos Kakkanas at the Hellenic Pasteur Institute. All team members, regardless of their primary roles, received thorough training in biosecurity. This comprehensive approach ensures that anyone within our team possesses the knowledge and competence needed to access laboratory areas as required. The seminar focused on the potential dangers when working in the laboratory, such as carcinogens, mutagens, highly flammable chemicals, acids and corrosive chemicals, cytotoxic reagents, neurotoxic chemicals, respiratory, skin and eye irritants, inflammatory reagents etc. Another part of the workshop concerned disinfection and sterilization. Going beyond theory, we transformed knowledge into action. Through a hands-on fire safety exercise, we used dry powder and carbon dioxide fire extinguishers. Each team member experienced the importance of swift, effective response, reinforcing our commitment to safety in all aspects.
The majority of our experiments were conducted at Professor A. Agathagelidis' laboratory at the Biology Department of the National Kapodistrian University of Athens, which is a BSL 1 standard laboratory. Some of our experiments were also hosted at other laboratories of the Biology department that provided us with the special equipment we needed. All of these laboratories are BSL1 standard laboratories.
In our laboratory, warning signs and safety instructions were hung on the walls. Also, there was a fire safety corner just outside the door. As indicated by the rules, there was never one person working in the laboratory alone, in case an emergency happened and they needed help.
All team members followed strictly and responsibly the dress code, which includes closed shoes, full-length trousers, and mandatory use of a lab coat, mask and nitrile gloves, not only when doing experiments, but also when spectating. We made sure that no food or drinks were exposed in the laboratory spaces by keeping them in a separate freezer in another room.
We were also very cautious with our waste management and we never discarded any living organisms or chemicals. Our used Petri dishes were all autoclaved before discarding, and all liquid cultures and consumables that were exposed to the microorganisms, such as tips, Eppendorfs and glass tubes, were incubated overnight with a strong chlorine/soap solution, before disposal.
When working with dangerous substances, in our case only ethidium bromide, we always worked on a separate special bench and used another pair of gloves. In the special case of UV exposure during Gel extraction, we also wore safety goggles and two sets of gloves and made sure that our lab coats covered our hands completely.
As the host organism we used was E.coli, disinfection played a very important role while conducting our experiments. An aseptic technique was always used and our microbiological work was done under flame. All the workstations were washed thoroughly and sterilized with 70% ethanol both prior to and after each experiment. In the most important or large scale experiments, we also had access to a safety cabinet in E. Pappa's laboratory.
Since we selected to work with a phage, we only used filtered pipette tips when handling M13 material, to avoid pipette contamination. We were also extremely careful when using common areas and machines, following the rules set by those in charge. Apart from our instructor's and professor's exhortations, combined with safety rules from bibliography, we decided to come in contact with someone who was working with phages in similar experiments. Nikos Moustakas, a BSc, ETE, working at Pasteur Institute, offered to provide important advice and indicated the stages of our experiments that required our utmost attention according to his own experience. He also visited our laboratory and demonstrated various manipulations of the M13KE phage in the laboratory.
Apart from implementing safety into our designs and using correct biosafety practices, those principles were also applied in our human practices and educational activities.
On September 27, we organized an event in the conference hall of the University of West Attica called “Biosafety through theater: From one mask to another”. This included a biosafety debate, continuing the tradition of the previous iGEM Athens team, with the subject of whether Biosafety regulations are reasonable or too restricting. An informative presentation on biosafety as a whole was shown, covering topics from correct biohazard disposal to kill switch design. Finally, the audience participated in several theatrical games on biosafety, with the intention of reminding and internalizing key topics and themes from the previous activities, and to leave them with a more personal and experiential impression of the event and its themes.
On several occasions , we performed a banana DNA extraction experiment with members of the general public. Even though the experiment was harmless, we emphasized on the importance of hand washing, surface cleaning and safe lab work practices in general. This also applied to workshops where we provided microscopes for children to use, encouraging them to clean the lenses and handle the material with care and to the events where we along with young children, created DNA's double helix by edible candy. We were persistent that everyone washes their hands before handling the candy, so they could safely eat them later.