Team:HZAU-China/Review

Our project includes three modules:an input module, a processing module and an output module. In order to test our design principle, we design two rewirable circuits. We build a repressilator with lox71 and lox66 to achieve the altering from oscillation to a stable switch. And we also construct another circuit that is related to quorum sensing to perform the transformation from the positive feedback to the negative feedback. To reduce the leakage of the cre expression, we add the riboregulator in our input module. And we have two plans to the output module because of the oscillator‘s quick response requirement. One is the fluorescent proteins with the Lva tag, the other is spinach RNA aptamer. We synthetized the crRNA by PCR by ourselves, and the RNA aptamer was synthetized by Tshing Ke Company. Most of our parts are constructed by BioBrick™. Due to the reverse construction in the processing modules, we design a new way based on the BioBrick™ to assemble those devices.

Furthermore, we found the unexpected recombination of lox71 site, and we designed the device and confirmed this phenomenon, then we tested if it would affect our results. And the answer is no. We did the promoter test about all inducible promoter we used by microplate reader. We also helped HUST-China to sequencing their promoters and did the promoter test. To test whether our riboregulator work, we replace the cre with the mCherry in the input module to detect the fluorescence intensity. Also we tried many times to synthesize the DMHBI but the purity cannot meet the experiment requirement. At last, Zhuang Shaoping senior helped us to synthesize it. Then we tested the Spinach RNA aptamer by fluorescence microscope to prove it can work. At last, we plan to test the processing modules by the fluorescence microscope and fluorescence spectrophotometer.