Team:NCTU Formosa/Experiments
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Overview
Integrating the designed gene circuit and model, we performed qualitative and quantitative analysis. What we have done, specifically, was to transform the toxin gene into E. coli BL21(DE3) and observed its growth curve. Based on the design of the toxin gene circuit and its selection logic, when the engineered E. coli encounters a growing environment with mutagen, the growth will be affected accordingly. After we documented the results from different situations and model analysis, we could thus build up a qualitative and quantitative analysis of mutagenic factors of interest.
The experiments consisted of two main parts. One was constructing the vectors with iGEM standard cloning, and the other was the functional test of biobricks.
In the experiment part, we first selected the toxin gene, which could steadily exist when it was not induced. Then we cloned five toxin genes into E. coli BL21(DE3), a strain that could produce LacI protein, respectively. So, the toxin gene would not express until we added IPTG for induction.
We ran the electrophoresis of Taq PCR products to verify that our target gene was correctly cloned into the E. coli, we carried out the functional test of each toxin gene. Then we chose the ccdB gene and the ydfD gene to perform the mutagen bioassay with two well-known mutagens that cause DNA damages, EtBr and UV light.
Altogether, we combined our experiment data points with model simulation to fit the growth curve and also calculated the mutation rate(M2) that quantified the mutagenic intensity and strength of the two mutagens.
Figure 1: The workflow of our experiment
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