Difference between revisions of "Team:ShanghaiFLS China/Safety"

Goal

We aim to maximize the methanol conversion rate and lower oxygen consumption and heat generation in Pichia pastoris metabolism. This may be achieved via 1. Refining the metabolic pathways of Pichia pastoris so that it may consume methanol and other carbon sources (such as glycerine) at the same time and 2. Converting metabolic byproducts (formic acid) back to methanol so that methanol may be utilized to the greatest extent.

Experiment

In the GS115 cell line of Pichia pastoris, there are three transcription factors for the AOX1 gene that encodes aldehyde oxidase (the protein that allows it to metabolize methanol), respectively Prm1, Mit1, and Mxr1.

We have re-engineered the metabolic pathways of Pichia pastoris GS115 to allow it to produce the same yield while consuming less methanol, hence lowering the oxygen consumption and heat generation. This may be achieved by regulating the expression level of Prm1 and Mit1 by interchanging their promoter sequences (Pprm1 and Pmit1).

The corresponding plasmids were constructed in E. coli Top 10 and then introduced into P. pastoris GS115. The engineered P. pastoris GS115 strains were then incubated in 24-well plates in media of different methanol concentrations. Samples were taken at regular time intervals, and the cell density and fluorescence intensity were measured in a microplate reader. Normalized fluorescence intensities for 1OD of cells were then calculated and analyzed.

Managing Risks

Our PIs are responsible for the safety and security of the biology labs. They have monitored our team members when we were conducting the experiments, to see whether we have strictly followed the lab protocols. They have also offered help whenever we needed assistance. Our PIs include Ph.D. candidates at the university lab that we borrowed, so they have a comprehensive understanding of Pichia pastoris, proper lab equipment, and lab protocols.

We have strictly confined the resistant cells and disposed of the culture in designated areas only. When conducting electrophoresis, we wore protective gloves and did not contaminate anything out of the electrophoresis room with anything inside.

We also used risk management tools to make sure the environment and our team members are safe. For example, we used accident reporting to record any accidents happened; personal protective equipment like lab coats, gloves, eye protection, etc; inventory control system to track who has what materials and where they are; medical surveillance to find out if our wet-lab team members get sick because of something we are using; special procedures or protocols that address safety or security.

During our training session, we learned the rules in the laboratory. For example, we learned that we should wear protective garments at all times when we were conducting the experiments. We also learned about the equipment in the lab. Since we did not have access to dangerous equipment like the autoclave, we were exposed to minimal danger. Also, since we were working in the bioengineering lab at East China University of Science and Technology, all the materials that we disposed of were processed before release.

Compliance with iGEM Rules and Policies

1. We have not released any organism or product derived from our project.
2. We have not used any animals (including insects and invertebrates) not on the Whitelist.
3. We have not used any vertebrates or higher-order invertebrates.
4. We have not used any parts not on the Whitelist.
5. We have not carried out any activities not on the Whitelist.
6. We have not used any parts or organisms obtained from outside the lab or regular suppliers. We have not released any organism or product derived from our project.

Conclusion

We strictly followed iGEM rules and safety policies, including thoroughly reading security rules and the Whitelist, providing iGEM with our safety information, and attending multiple training sessions. Overall, all of the information provided by iGEM was very useful.