Team:Worldshaper-XSHS/design.html
Design
Based on our previous study, interviews and social investigation results, we planned to develop an efficient, affordable and convenient method to monitor the concentration of nicotine in the particular environment. If the idea of the experiment is proved feasible, the experimental results are likely to be directly applied into the environmental quality testing of relevant units. The first step in this plan is to construct plasmids that can express specifically in the presence of nicotine. After comprehending a great number of related researches and reading a plenty of related literatures, we finally found the Nox and NicA genes that bear on nicotine degradation, and predicted their promoters by literature【1】 and several websites. Then we named these promoters Pnox, Pnica1 and Pnica2 We hoped their promoters to be nicotine-induced and if fortunately the result is exactly as we expected, it would be possible for us to use the synthetic biological methods to construct E. coli strains as a component of detector.
Design of the First Version
First of all, we designed the first version of the nicotine induced gene fragment, including: the predicted Nox promoter as a sensor, and the GFP gene as a reporter. In a single cell, different amounts of GFP will reflect nicotine concentrations in different nicotine solutions. We hope that nicotine concentration will be reflected in different fluorescence intensity eventually. Unfortunately, the part contains GFP gene could not be successfully transformed into E. coli, so our following experiment was shelved. Later we used RFP and BFP gene instead of GFP to reflect nicotine concentration. However, RFP gene doesn't contain RBS and terminator while BFP gene doesn't contain terminator, and may need later addition.
We found that the Nox promoter was not a nicotine specifically induced promoter, so we predicted and built two series of NicA promoters. At the same time, we were lucky to borrow the GFP gene transformed Escherichia coli strain, so that we designed two of the first version monitoring systems that using NicA promoter as the monitor, GFP gene as the reporter and containing the double terminator. The result shows that one sort of predicted NicA promoters could be specifically induced by nicotine.
With these three plasmids construction experiments, we hope to further improve the expression efficiency of GFP on the basis of the first generation detection system, so that we can more accurately detect nicotine concentration in the environment. With the help of Professor Zhu Xufen, we use T7 RNA polymerase gene and T7 strong promoter to improve the efficiency of GFP expression. Therefore, we hope that the gene fragments of the second generation detection system include: Pnica2 promoter, T7 RNA polymerase gene, T7 strong promoter and GFP coding gene. Pnica2 promoter does not directly control the expression of GFP, but directly controls the expression of T7RNA polymerase. The latter was consistent with the T 7 promoter and the expression efficiency was much higher than that of the other combinations. We expect that GFP, indirectly regulated by Pnica2 promoter, will have higher expression efficiency. Of course, later experimental results confirm that our expectation has been fulfilled.
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