Safety
Since we are working with genetically modified organisms, the safety of the lab workers and the environment was paramount. Throughout our journey, we actively communicated with safety advisors and eventually put useful approaches into practice. We used all the received advice to shape our projects and think about precautions that are crucial to fulfill the claims of biosafety: the lab environment needed to be safe, and we had to put in place a protocol to protect the outside environment in case our strain got outside the lab.
Work Environment and Equipment
We put in place several safety guidelines for the general lab environment and handling of our engineered strains.
Designated Area: To prevent contamination within the lab, we restricted all work with genetically engineered Chlamydomonas to designated areas (biosafety cabinet).
Sterile Work Area: We ensured a sterile environment for procedures by cleaning surfaces with a disinfectant (e.g., 70% ethanol or a bleach solution) before and after the experiment.
Autoclave:
All waste and materials were autoclaved to sterilize them and prevent environmental release. We transported genetically engineered Chlamydomonas strains in sealed, leak-proof containers. We also added secondary containment, like sealed plastic bags or rigid containers, to reduce the risk of accidental release. To ensure all was accounted for, we documented all experimental procedures, strain details, and disposal methods in our lab notebook.
Kill Switch
A kill switch is a system that kills our engineered organisms if it spreads in the environment, providing security in case of leaks and catastrophes. The conditions in the lab and in the environment differ, hence the possibility of acting on this difference to kill our organism if the conditions of culture change, as it is the case if it leaks in the environment.
A kill switch for E. coli
We have found a kill switch described by other IGEM teams which involves the detection of blue light by a known photoreceptor. (USTC 2019, Unesp Brazil 2018)
It is a photoreceptor named VVD, isolated from Neurospora crassa. It is composed of 2 parts that dimerize under blue light. We divide Cas9 gene in two parts, that we link to each of the two photoreceptor parts. As the blue light reaches the photoreceptor parts, everything dimerizes and Cas9 protein is formed.
We also add in the bacteria some guide RNA (gRNA) that will suppress the expression of some essential genes, inducing the death of the bacteria.
A kill switch for C. reinhardtii
We reuse a kill switch that has been found by the Sorbonne U 2020 team. This system is also based on light, but this time UV light is involved. In the lab, the algae is not exposed to UV light, however it is exposed to it in the environment.
A nuclease from Staphylococcus aureus is produced with a NLS (nucleus localisation signal) and a transmembrane domain. In normal conditions, the nuclease is localized at the cellular membrane, and it cannot act on its target, the DNA in the nucleus.
A protease expressed only with a UV light trigger is engineered. Like with Cas9 and the photoreceptor for E. coli, the protease is cut in two and each part is linked with a UV light receptor that dimerizes after being triggered by UV rays. The protease, which is the TEV protease, is then assembled and able to cut the nuclease at a specific site, which separates the transmembrane domain from the remaining nuclease. The nuclease is then able to relocalize in the nucleus and cut the DNA. The fragmentation of the DNA induces cell death.
The constructions are the N-terminal part of the TEV protease linked to COP1 and its C-terminal part linked to UVR-8.
The nuclease may not come from the algae but it has been demonstrated that it works well (Tilbrook et al. 2016).
We have not tried those systems in our experiments because of a lack of time. If the project is continued by other teams or industries, it will be necessary to add those systems in the organisms.
References
https://2018.igem.org/Team:Unesp_Brazil/Design