Team:HZAU-China/Review

From 2014.igem.org

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         <p class="highlighttext">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. </p>
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         <p class="highlighttext">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 site and lox66 site to achieve the alteration from the state of oscillation to the state of a stable switch; and we also construct another circuit that 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, we add a riboregulator in our input module. As to our output module, we get two devices to detect whether our processing system works or not. One is the fluorescent proteins with Lva tag, the other is the spinach RNA aptamer. We synthetize the crRNA by ourselves, and the RNA aptamer is 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. </p>
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<p class="highlighttext">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.</p>
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<p class="highlighttext">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.</p>
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Revision as of 07:37, 16 October 2014

<!DOCTYPE html> 2014HZAU-China

Overview

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 site and lox66 site to achieve the alteration from the state of oscillation to the state of a stable switch; and we also construct another circuit that 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, we add a riboregulator in our input module. As to our output module, we get two devices to detect whether our processing system works or not. One is the fluorescent proteins with Lva tag, the other is the spinach RNA aptamer. We synthetize the crRNA by ourselves, and the RNA aptamer is 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 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.

Contacts
  • No.1, Shizishan Street, Hongshan District
    Wuhan, Hubei Province
    430070 P.R.China
  • Wechat : hzauigem
  • QQ Group : 313297095
  • YouTube : hzauigem