Since one of our core values was (bio)safety, we took all safety issues into account in every phase of our project. That included safety evaluations of laboratory work as well as safety regarding COVID-19 and our proposed implementation. In addition, we considered some bioethics aspects.
According to iGEM’s safety rules, we filled the safety form in which we carefully and diversely discussed all the safety issues regarding our laboratory work. Naturally, we also obeyed all international, regional, national, local and institutional regulations. Our work didn’t include working with animals or humans. This means that all parts used and activities done in our project, were on the iGEM whitelist. In addition to this, specific rules and legislations that we obeyed are mentioned in our proposed implementation page.
At the beginning of our project, all our laboratory personnel got a tour in the laboratory facilities and all the safety procedures were repeated and safety training conducted. The biosafety level of our laboratory was 1 and all of our laboratory facilities had a GMO-permit that is required by national legislation. When working with our experiments, at least one other person from the molecular plant biology or biochemistry unit of our university had to be present. All in all, the lab we were working in, had comprehensive safety and security plans.
In our project, we were using commercially available Escherichia coli laboratory strains DH5α and BL21(DE3). The E. coli DH5α strain contains recA1 and EndA mutations to increase insert stability, and DNA yield and quality, respectively (Chen at al., 2018). The E. coli BL21(DE3) strain, in turn, lacks the proteases Ion and ompT and contains the prophage DE3 from the bacteriophage lambda (Jeong et al., 2015). These strains are not pathogenic to humans and they are domesticated laboratory strains so it’s unlikely that they would survive competition outside the laboratory. To ensure no bacteria would get outside the lab, we inactivated all the bacteria after use and we also inactivated the bacteria containing trash.
The expression plasmids (pET36b carrying the laccase genes cotA, cueO and yak) are not linked to any harmful characteristics. However, the plasmids contain antibiotic resistance genes which can in theory be transferred to other microbes. Thus, it’s important to prevent their spreading into the surroundings. One of the laccase genes we were trying to express, yak, is originally from Yersinia enterocolitica subsp. palearctica strain 7. That organism is in the risk group 2 and it has been defined as a zoonotic pathogen to humans, causing a disease called yersiniosis (National Institute of Health, Centers for Disease Control and Prevention). However, we weren’t using the source organism itself, but ordered the yak gene through gene synthesis. The yak gene itself is not associated with any harmful consequences.
We also used in our project some chemicals that can in theory be potentially hazardous. These reagents included hydrochloric acid, methanol, ethanol, redox agents 2,2'-(1,2-hydrazinediylidene)bis[3-ethyl-2,3-dihydro-6-benzothiazolesulfonic acid and diammonium salt, as well as potential mutagen SyBR Safe DNA Gel Stain by Invitrogen. With these chemicals, and also with other typical laboratory chemicals, we paid extra attention to safe handling and general caution. When working with potentially hazardous chemicals and wastewater samples, we performed our experiments in chemical fume hoods. All material and waste resulting from our experiments was disposed of according to standard laboratory and GMO regulations.
After having stated all of the safety measures above, we’re also going to mention that we also took into account the very basic safety measures in a lab environment. We used personal protective equipment such as lab coats, nitrile gloves and safety goggles and we never drank or ate in the lab. In addition, we made sure that we would report all accidents that might happen in the lab.
Naturally, we had to take the ongoing Covid-19 pandemic into account throughout the iGEM year. We obeyed the national restrictions and other recommended policies. We wore masks and kept safety distances to each other. We organized our regular team meetings remotely for the most time of the year, even though we were able to meet in person this autumn. It was also obvious that in the case of being sick, we stayed home.
Bioethics & Biocontainment
We’ve considered bioethics in our project with the help of these following questions:
- Why do you deserve public trust?
- Are you presenting your findings clearly and in a reproducible way?
- How will your research benefit society?
- How might someone misuse your technology?
We believe that we deserve the public’s trust because we’ve taken extra caution in every step of the way of the project. We’ve double-checked and we’ve done comprehensive research on literature. We’ve also reported everything in our wiki in a reproducible and reachable way. In addition, our project has also been about bettering the well-being of our society. This is because helping the Baltic Sea will eventually result in for example maintaining the possibilities of relaxation in the sea for the general public.
We also considered what would happen if our constructs would be misused and how we could prevent that. We had to consider this possibility if someone were to get their hands in our drug-degrading laccases and find a way to use them in an unethical way. For example, they might have used them to degrade drugs and sell them to gain financial benefit. This would however be prevented from happening by following the national rules and legislations of GMO handling. If these safety measures are followed well enough, no random people should get in touch with our system.
In addition, we obey iGEM’s Do Not Release Policy which states some basic principles of how to be a responsible scientist and avoid releasing GMOs to the environment. Our proposed implementation plans include producing laccases by Synechocystis sp. PCC 6803 in a closed bioreactor system as a part of a wastewater treatment plant. The escape of genetically modified organisms is trying to be prevented in multiple ways. From a closed bioreactor system, only laccases produced by cyanobacteria would flow through the filter to the next container. We are planning to integrate an effective metal-incudible killswitch, for example MazF, to the genome of cyanobacteria. Our technical solution would also include a separate kill tank, in which the killswitch is activated upon accidental release of the engineered cyanobacteria.
Read more about our proposed implementation from here.
- Centers for Disease Control and Prevention. Yersinia enterocolitica (Yersiniosis). Retrieved Oct 19, from https://www.cdc.gov/yersinia/index.html.
- Chen, J., Li, Y., Zhang, K., Wang, H. (2018). Whole-Genome Sequence of Phage-Resistant Strain Escherichia coli DH5α. Genome announcements, 6(10), e00097, https://doi.org/10.1128/genomeA.00097-18.
- Jeong, H., Kim, H. J., & Lee, S. J. (2015). Complete Genome Sequence of Escherichia coli Strain BL21. Genome announcements, 3(2), e00134-15, https://doi.org/10.1128/genomeA.00134-15.
- National Institute of Health. Guidelines. Retrieved Oct 19, from https://osp.od.nih.gov/biotechnology/nih-guidelines/.