Designed Protein Degradation Method Based on
Trim21 And Nanobody -- Safety
In our project, we use engineered E.coli and plasmids for the molecular cloning which aimed to build the gene circuit for the expression of Trim21 and the recombinant antibody. To express Trim21 and the recombinant antibody in mammalian cells, the gene circuit were integrated into pcDNA3.1 vector and were transiently transfected into Hela Cell or HEK293T Cell, to prove the concept and further demonstrate our project respectively. Western blotting and cell proliferation assay were used to evaluate the function of the designed gene circuit. The cells, plasmids, as well as other materials we used in our project were all commercially available, which were widely considered to be safe for us to work with. The technique used in our experiment, such as molecular cloning and Western blotting and cell proliferation assay, were all routine methods. The gene circuit we built was a combination of pre-existing gene fragment which were reported in public literatures. The new achieved property of the gene circuit was to strengthen the specific cell function with high specificity, which were realize by the specific binding to target protein by recombinant antibody. Thus, our gene circuits woubld not introduce any threat to nature. To date, our experiments were performed on cell level, no viral vector, animal or clinical experiment were involved. Several concerns of the safety of our project as well as the laboratory works are addressed below.
1、We choose genetically modified E.coli as our chassis organism in our project. Our chassis bacteria have not acquired any characteristics that would enable them to compromise human immune system/other systems or evade detection and destruction by the former or facilitate spread between people/animals, which makes them harmless from both a personal and public health point of view.
2、Our parts are all safe for human, we ensure that none of the genes or parts that we were assembling would act as virulence factors, and that no known pathogens would be involved in our research.
3、All genes were cloned into either pSB1C3, puc57 or pcdna3.1, all of which are non-conjugative, preventing horizontal transfer of our parts. We use LacI as promoter, which guarantees that our bacteria won’t produce large amounts of protein if not induce the expression.
Safety Lab Work
1、Before we start our experiments, our advisors gave us a wonderful lesson about Lab safety. From that, we have learned a complete understanding of experimental risks associated with synthetic biology.
2、We strictly follow the guideline of the WHO lab biosafety manual and the instruction of our instructors. We are required to understand the experimental principle and method completely before the operation.
3、As for electrical safety, the electrical engineer of the lab showed us the circuit layout and told us details about the use of these equipment. When finish the whole experiment that day, we take turns to check these electric appliances. Before using every kind of equipment, we read the instructions over and over again to avoid any safety problems, and we cut off the power immediately when do not use them. Particularly for using centrifugal machine, it is significant to make sure that you have already balanced it and covered the lid well before centrifugation.
4、All wastes are sterilized with proper treatment and finally send to qualified companies. The abandoned bacteria can only be thrown away after high temperature and high pressure sterilization.
5、We are enforced to wear gloves, masks and lab coat once we enter our lab. It is prohibited to bring anything in the lab out including gloves, masks and lab coat. Therefore each time we get out of the lab, we have to take off lab coat, gloves and wash hands with hand sanitizer.
6、When using the lentivirus pLV and TG006, we carefully follow the safety direction.
7、Inevitably, we are exposed to toxic chemical reagents, such as GelRed, DEPC and so on, the possible expose to UV light may also raise safety concerns. To reduce such risks, separated area were defined and extra protection were performed when operation toxic chemical reagents and UV lights. We put the toxic chemical reagents in the fume cupboard, any relevant operations are all done in the fume cupboard, and we separate a special area for the preparation of agarose gel and agarose gel electrophoresis, we need to wear two pair of gloves to touch anything in this area.
As mentioned above, our DNA parts are absolutely safe. The DNA parts are safely dried down within 96 well plates, sealed completely, and covered with a protective lid as Parts registry requires.