Safety in Project Design
In our project, we demonstrate a designer cell approach to control hyperglycemia by degrading hepatic Glucagon Receptors (GCGR) in a glycemic dependent manner. We achieved glucose responsiveness by synthetic circuits that couple endogenous CHREBP glucose sensing pathway to a CHREBP activating hybrid promoter controlling GCGR degrading elements. The degradation of GCGR was then executed by proteasome-based system in a Trim21 based, Trim-away alike manner. Circuit-carrying HepG2 cells showed significantly decreased GCGR level and glucogenic ability, similar results were obtained in primary mouse hepatocytes with adenovirus as delivery approach as well. And the following safety issues have been considered in our project design.
1. Adenovirus was used for circuit delivery in our experiment. We reached out Safety hub for instructions and permissions which said our ideas are feasible before we conduct our experiment (Figure1) and consulted experts and other iGEM teams for suggestions. By their suggestions and approval from both iGEM Safety committee and our department safety committee, we performed following extra precautions when conducting our experiment: Replication defective virus was used to make it unable to replicate in the natural environment. In addition, secondary biological cabinets (Figure2) were used to enhance the safety of the experiment. We also conducted collaboration with CSU_CHINA to try other non-virus approaches.
2. All parts we use are basically safe and will not cause any biological pollution to the natural environment.
Figure1:reached out Safety hub
Figure2:secondary biological cabinets
Safety in lab work
1. All our operations comply with the safety regulations of the laboratory safety manual.
2. Before each team members starts the experiment, our advisors gave us a wonderful lesson about Lab safety, and the old members will help and guide the new members to avoid mistakes in the operation. Here is the emphasis on the specification:
- Wear white coats, masks and rubber gloves.
- Read the experiment procedures carefully before starting the experiment and get familiar with the operation steps; Be familiar with the function of each experimental equipment；Prepare all the equipment that will be used in the experiment, avoid looking for experimental equipment in the experiment.
- Reasonable and scientific treatment of experimental waste is needed to prevent pollution.
- Standardize experimental operation and avoid wrong operation.
- Calm down and learn to deal with some possible accidents.
- We can’t operator alone. Besides the operator, there will be teammates to observe and correct the possible wrong operation in time(Figure 3).
3. 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.
4. After the experiment, the experimental personnel will clean up all kinds of experimental equipment and waste liquid which were generated by the experiment on the operation table in a unified and safe way to prevent possible harmful bacteria from polluting the environment.
Figure 3:observe and correct the possible wrong operation