Team:HZAU-China/Review

Our project includes three modules: an input module, a processing module and an output module. In order to realize the principle of our design, we design two rewirable circuits: a repressilator with lox71 site and lox66 site to achieve the alteration from the state of oscillation to the state of a stable switch; and another rewirable circuit which is related to quorum sensing to perform the change from positive feedback to negative feedback. To prevent the leakage of the expression of Cre protein in our input module, we add a riboregulator in front of the coding sequence of Cre protein. As to our output module, we get two devices to detect whether our processing module works or not: one is the fluorescent proteins with Lva tag; the other is the spinach RNA aptamer. Most of our parts are constructed by BioBrick™,but for purpose of meeting the special requirement of our construction that we need to connect some parts reversely, we design a new way based on the BioBrick™ to assemble those circuits.

Furthermore, we found the unexpected recombination of lox71 site, and we designed a device to verify this phenomenon, and then we tested it if it would affect our results to some extent, and the answer was NO. We also conducted a experiment for all these inducible promoters we used in our gene circuits by microplate reader to get some information about their intensity which may help us to select the optimum promoter. Definitely, We did help HUST-China to sequencing their promoters and did this promoter test. To test whether our riboregulator works or not, we replaced the coding sequence of Cre protein with the coding sequence of mCherry protein in the input module to detect the efficiency of this riboregulator by fluorescence intensity. Also we tried many times to synthesize the DMHBI but the purity still couldn't meet the experiment requirement and so, we found a senior called Zhuang Shaoping to help 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.