Safety Project Design
All our work is covered by the White List. In our research, we used E.coli TOP10 cells for plasmid generation which are non-pathogenic and stable. Safety concerns are mainly regarding the bacteria we are manipulating.The genetic modifications we introduced do not increase their dangerousness. The entire process of our work is conducted in academic laboratories and all team members have received safety training. Appropriate and required protection was used during all steps of wet lab manipulations.
Safe Lab Work
General safety
Before starting any experiments, our team members were trained on safe handling practices, biosafety protocols, and security measures to prevent accidental release or misuse of the technology. Moreover, the iGEM mentor, who is paired with our team, as well as our PI, supports us in managing risks associated with our project. We are also staying updated with iGEM's guidelines. There are instances where animal experiments or experiments with plant species would be more effective, but we considered that the risks outweigh the benefits and we decided to go against conducting such activities to prioritize ethical considerations and biosafety. We have developed a communication plan that responsibly addresses the information shared about our project. This involves redacting specific details that could pose security risks or potential misuse, while highlighting the biosafety measures implemented to ensure responsible and ethical research.
We adhered to established rules and guidelines, such as those provided by iGEM, regulatory authorities, and ethical standards, which helped us to ensure that our project is conducted in a responsible and compliant manner. These rules provided us a framework for risk assessment, containment, and safety protocols. Our team members were trained on biosafety practices, security measures that are important for risk management, and equipped our team with the knowledge and skills necessary to handle the project safely. Training sessions covered topics such as laboratory safety, containment procedures, waste management, and emergency protocols.
We also have access to well-equipped laboratories and containment facilities that meet the necessary biosafety level requirements, which is essential for managing the project risks.
Properly maintained equipment, such as biosafety cabinets and waste disposal systems, ensures the safe handling and storage, minimizing the risks of accidental release or exposure.
By following these rules, receiving expert support, undergoing training, accessing appropriate equipment and spaces and adhering to protocols, our team effectively manages the various risks.
Biological safety
Bacteria Strains and plasmids
For our research, we used chemically competent E.coli TOP10 cells for plasmid generation, The plasmids we used are in the table plasmid and parts (obtained from iGEM kit plate, more details have been registered in the Experiment wiki page). The appropriate antibiotic used in the experiment: 25 μg/mL chloramphenicol (Cm) 100 μg/mL ampicillin (Amp), 50 μg/mL kanamycin. All experiments related to prokaryotic cell transfection are carried out on the ultra-clean bench of the prokaryotic cell lab, which ensures that the experimental process is safe, hygienic and free of contamination.
Vectors
For our project, we used two different vectors, of which detailed information can be found on the Experiment wiki page. Vectors that we used for sequencing are pSB1C5C for Nickases Cas9n D10A, H840A, Cas_varient_dhSpCas9, Cas variants hSpCas9 and pSB1C3 for 3_Colors_ins_K2656022 according to the iGEM's public material library. We sent the plasmids to Sangon Biotech for sequencing, this professional biological company can ensure that the sequencing process is safe and harmless. All the necessary primers and oligonucleotides were also ordered in Sangon Biotech.
Laboratory equipment
In our laboratories, ultra-clean benches are cleaned with alcohol and UV light after use, and reusable laboratory equipment are thoroughly cleaned and autoclaved after use. We have specific areas to separate "dirty" materials and equipment, from "clean" to prevent contamination. Laboratory waste is sorted and, in particular, hazardous waste is specially handled and kept separately to avoid contamination.