Before the Lab Work
While still planning our project and figuring out finer details, our PI, Dr. Nicole Gensch, provided us with thorough safety instruction according to our university’s standards for working safely. For more information about our university’s safety standards, click here.
She explained key points and main elements regarding working in the lab safely, including waste management, basic emergency plans, escape routes, and knowledge of the whereabouts of provided safety equipment like fire extinguishers and first aid kits. Additionally, she taught us the proper handling of chemicals and genetically modified organisms. We received the necessary training and required instructions to work on our own in a level 1 rated biosafety lab.
The First Week in the Lab
As our team consists entirely of bachelor students, our initial lab experiences at the beginning of our iGEM journey were limited. To bridge this gap, one of our instructors, Anne Smedegaard, generously took it upon herself to introduce us to practical lab work. She planned and provided instructions for a few basic experiments that all team members would eventually perform on a daily basis.
These experiments included simple PCR, gel electrophoresis, gel extraction, Gibson assembly, and the transformation of the prepared plasmid into E. coli. Having the opportunity to try these vital experiments for the first time under the guidance of someone experienced and knowledgeable about these processes was incredibly helpful for us. This approach allowed us to make our first mistakes under supervision, rather than later when working alone.
Working in the Lab
As mentioned earlier, our lab is equipped with an assortment of equipment for personal and general safety. In addition to the already listed general equipment aimed at minimizing personal risks and potential health concerns, personal safety gear such as lab coats, safety goggles, and gloves was provided and had to be worn at all times in our facility.
Throughout all experiments, we consistently sought input from our PI and supervisors/instructors on how to minimize potential threats, ensuring our lab work was done in the safest way possible. In the early stages of our lab work, one of our supervisors was present during every experiment, ensuring we knew how to handle and carry out the planned experiments. As we gained more experience, we worked more independently, but always had the benefit of having solid advice just a phone call away.
For each new machine (e.g., plate reader) and every new technique (e.g., Western Blot) that we planned to use in our experiments, we received specific instructions on how to handle them properly.
Our project was deliberately designed to never exceed biosafety level 1, as the lab where we worked most of the time only provided this specific level.
All experiments that were performed in connection to our project were planned in advance and discussed with our PI and supervisors to ensure their necessity and safe execution.
Given that our work involves a potentially harmful compound, a bacterial toxin, all experiments incorporating the MazF gene are carried out with an inducible promoter. This allows for precise control over the expression of the toxin, as it is only activated in the presence of doxycycline. Apart from this aspect of our system, all other components can be considered harmless.
For our experiments, we only used model strains of bacteria specifically designed for lab work, which are present on the whitelist and did not need specific permission from an ethics committee or special containment.
We took great care to ensure that none of our genetically modified organisms ever left the laboratory. Even if such an occurrence were to happen, considering that all strains were engineered for laboratory work, they would not survive in a natural environment.
Our autoregulatory system was designed to be universally applicable, allowing for a broad range of applications by simply exchanging one part of the system. As scientists, it is our responsibility to consider potential dangers, abuses, as well as the benefits of our creations. We developed CELLECT to enhance the production of specific compounds. By replacing the gene of interest, theoretically, any other molecule or harmful substance could be produced, limited only by the existence of a suitable riboswitch. The survival of cells can be controlled in a product-specific manner by replacing the riboswitch. It's important to note that most compounds dangerous to humans would likely also harm the host, leading to the cessation or reduction of the production of that molecule.
The same principle applies to the second use intended for CELLECT: degradation. When a substance is degraded into a more harmful product, potential harm can occur. However, we must consider that bacteria can adapt to their environment. Therefore, they would either suffer damage by producing harmful compounds or die due to the function of CELLECT.
However, another potential risk that needs to be addressed is the possibility of mutation of the toxin. In this case, bacteria that are able to degrade and potentially utilise otherwise harmful substrates have a clear advantage over wild-type bacteria. Therefore, they outgrow wild-type bacteria and cause unknown damage and problems in nature.
Real Life Application
CELLECT is mainly designed for application in a bioreactor to scale up production. This ensures closed containment, eliminating any risk of release into the environment. Products can be secreted into the supernatant or extracted from the cells for further use.
During our project, we furthermore explored cyanobacteria as a chassis for our system. Another potential use of our system could be B12 production in cyanobacteria for direct consumption as a supplement. However, not all strains of cyanobacteria are currently considered "safe for food". Beyond the safety regarding intake of cyanobacteria the same safety concerns of contamination of natural environments as described above would apply.
For application in terms of degradation, the genetically modified organisms (GMOs) would need to be released from containment or the bioreactor, as the main idea behind this version of our system is the use in case of oil spills or other environmental pollution. However, the advantage of CELLECT is that the GMOs would die as soon as they finished degrading the compounds and would therefore be unable to spread into the environment.