In the hope of getting further support and help from other teams, our United_Shanghai_HS team and ASTWS_China team held an exchange meeting on the Internet via video on September 18, 2021. The guests present are the core members of the two teams.
First of all, Xiang Ruixi from our team introduced the background of our project, experimental design, and problems encountered to the participants. Then, our classmate Li Zexuan added. In response to our project, the other party also put forward many questions and suggestions.
About plasmid construction:
In the original plasmid construction, we initially only used the genes ISF6_4831 (PETase encoding gene), ISF6_0224 (MHETase encoding gene), (GFP fluorescent protein), but this design will reduce the subsequent sucrose amplification effect, and the CPU team proposed that With the addition of the reporter gene mCherry, they believe that when the transformed plasmid Bacillus subtilis is not strictly regulated by the sacB promoter, some ethylene glycol will be initially degraded. With the degradation of a certain amount of ethylene glycol, B. Glycol will hide the terminator contained in the riboswitch, and MHETase will be expressed normally. At this time, mcherry as a reporter gene will prompt the need to add sucrose amplification effect, there will be more ethylene glycol to enhance the transcription of MHETase. Present a positive feedback effect.
Regarding the design of the suicide switch:
The other party asked whether it is necessary to design a component similar to a suicide switch, because it involves the biological safety of the release into the soil, and we responded: Natural Bacillus subtilis can be discharged into the soil as a probiotic, in modern agriculture The application is very wide. The protein we use is also selected from the natural flora, and it is not artificially modified to have an impact on the environment. Generally speaking, the biological safety is relatively high. Therefore, the design of suicide genes was not considered at the beginning.