Though using synthetic biology to engineer microorganisms has been a common and well-researched approach to biotechnology, the importance of safety still needs to be addressed. Especially when we decided to use one of the genome editing tools, S. pyogenes (Sp) Cas9 endonuclease, to manage biological spills and leakage is our primary safety concern. Besides, we pay attention to safe experiment design and laboratory practices to ensure the safety of our project and the safety of our members.

During the construction of Chlipid, we used Chlamydomonas reinhardtii (Strain CC-503) as chassis and Escherichia coli (TreliefTM5-alpha, mutant strain of DH5-alpha) to construct CRISPR plasmids, both of which are commonly used in the lab and on the white list. After consulting our PIs, we chose SpCas9 endonuclease as the genome editing tool which is potentially hazardous, therefore, we take biological leakage as the main safety issue that needs to be prevented.

In order to prevent leakage of engineered organisms, we strictly perform sterilization before disposing of any engineered organisms in the lab. We also collaborated with team Sorbonne_U_Paris to design a kill switch to secure the process. This kill switch was inspired by 2020's Sorbonne University iGEM team, who were themselves inspired by Munich's 2013 iGEM team. They wanted to address Nuclease A to the plasma membrane by linking it to a transmembranary domain and ER translocation signal peptide. In between the nuclease and transmembranary domain would be a TEV-specific cleavage site. Therefore, under escape conditions, UV-sensitive dimerization could assemble both parts of the TEV protease, liberating the Nuclease A, which would then localise to the nucleus and provoke cell death(Figure 1).

In order to make sure all laboratory practices would be safe and under control, all of the members received safety and security training from our school's biological safety department. And all the wet lab team members take the Synthetic biology training course taught by our PI. We studied the basic molecular biology experiment's operation and safety precautions in the series of lessons. If we discover hazards or risks, we seek help from the schools' biological safety department, PIs, and their PhD students. All actions in the lab follow Biosafety Law of the People's Republic of China and Laboratory safety management methods of the University of Electronic Science and Technology. We strictly perform waste treatment procedures to control the leakage of hazardous chemicals, parts, and organisms from the lab.