Team:Worldshaper-XSHS/design.html
Design
Based on our previous study, we planned to create an efficient method to monitor the concentration of nicotine in the particular environment. If the idea of the experiment is proved feasible, its 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 the results conform to our 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 with several gene fragments, including: the predicted Nox promoter as a sensor, and the GFP gene as a reporter. In a single cell, different amounts of GFP can reflect diverse nicotine concentrations in different nicotine solutions. We hope that nicotine concentration will be reflected
by observing different fluorescence intensity. Unfortunately, the part contains GFP gene could not be successfully transformed into E. coli, so our following experiment was ceased. 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, so it needs later addition.
Design of the Second Version
We found that the Nox promoter was not a nicotine- induced promoter, so we 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 could design two of the first version monitoring systems: using NicA promoter as the monitor, GFP gene as the reporter and containing the double terminator. The result showed that one sort of predicted NicA promoters could be specifically induced by nicotine.
With these three plasmids construction experiments, we expected to further strengthen the expression efficiency of GFP on the basis of the first generation detection system in order to accurately detect nicotine concentration in the environment. With the help of Professor Zhu Xufen, we used 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 did not directly control the expression of GFP, but directly controlled 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 had higher expression efficiency. Of course, later experimental results verified that our expectation has been fulfilled.
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