Team:NCTU Formosa/Future Prospect
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Introduction
QS Gene
Composite Parts
Future Prospect
Introduction
In the future, we intend to improve the project design to help people compare mutation rate of the different mutagens more easily and directly. So, we designed biobricks integrating quorum sensing system(Figure 1). According to our design, the higher the mutation potential is, the faster the E. coli population will reach the threshold and begin to express red fluorescence.
Figure 1: Quorum Sensing Regulated RFP System. LuxI and LuxR are two important QS regulation genes in E. coli. LuxI protein catalyze the synthesis of AHL, which conjugated with LuxR to form a operator that promotes pLux regulated downstream expression. RFP will be expressed once the E. coli population proliferates, which also conform with the logic of growth curve selection.
To accomplish the idea of visualization, we have already started the preliminary experiment. First, we cloned the quorum sensing system into the E. coli and conducted the functional test. Then, we co-transformed them with toxin genes. In future experiments, we will do functional tests of the co-transformed E. coli. After that, we will put the co-transformed E. coli together with different mutagens and accomplish furthermore progress to our project.
Quorum Sensing Gene
Cloning
The Figure 2 is the electrophoresis results of the PCR products with 1Kb marker on the left side and target gene on the right side. The lengths are labeled beside each band.
Figure 2: DNA electrophoresis of part BBa_K3256437 with BBa_K3256439.
As the figure we show above, we successfully cloned QS gene into the E. coli.
Functional Test
Then, we tested the function by measuring the fluorescence intensity and the optical density of the transformed E. coli together with controlled group. We put our sample into the CLARIOstar® Plus plate reader. For each sample, we applied triple replication in order to get more precise data.
Figure 3: EtBr mutagenic effect on E. coli growth with quorum sensing rfp expression
In the beginning, the fluorescence intensity remained relatively at low level. At about 150 minutes after IPTG induction, the fluorescence increased sharply and then remained in high intensity. The result was in accordance with our quorum sensing design. The sharp increase of fluorescence intensity occurred at O.D. value at approximately 0.2, which was expected to be too early. This phenomenon was observed in multiple tests. We expected that the promoter Plux was loose and expressed well with little concentration of AHL. For future work of integrating Quorum Sensing system in the bioassay, we may have to change the promoter or add operon for the tighter control.
Composite Parts
Cloning
After we had done the function test of toxin gene and the QS gene circuit, we co-transformed the two vectors into E. coli BL21(DE3).
Figure 3 was the electrophoresis results of the PCR products with the marker on the left side and target gene on the right side. The lengths are labeled beside each band.
Figure 4: A. DNA electrophoresis of part BBa_K3256440 and BBa_K3256437 with BBa_K3256439. B. DNA electrophoresis of part BBa_K3256443 and BBa_K3256437 with BBa_K3256439.
We successfully co-transformed two different toxin genes respectively with QS gene into the E. coli. So, we will use them to do the following experiments.
