Team:BJU China/Improvement
IMprovement
Description
In our project, we constructed improved part BBa_K3887008.
T7 promoter is commonly used in iGEM projects, BBa_K2406020 designed by iGEM17_Edinburgh_UG was an inducile T7-LacO promoter for gene expression. In our project, we also used T7-LacO promoter to control the expression of halogenase. However, we found high basic leakage of this promoter, which affected the results of our experiments, so we improved this promoter in our project.
It is recognized that an excellent promoter should possess exceptional switch ratio: it should have lower background leakage in the "off" state and have obvious expression in the "on" state. In order to reduce the leakage and to ensure the repressor can bind better to the LacO site, we added another LacO site in the T7-LacO promoter as the figure below shows:
Fig.1 Improved promoter and original promoter
Gene Circuit and Test Method
In the experiment, LacI was designed as a repressor and IPTG as an inducer to control the state of T7 promoter. The gene circuit diagram is shown in Fig. 2. At the same time, we use sfGFP as reporter gene, so that the fluorescence value can be used to quantitatified gene expression level.
Fig.2 Gene circuit for promoter test
Single colonies of two bacteria (control, improved) were inoculated into LB medium, and cultivated vernight at 37° as seed culture. The seed culture was inoculated into M9 medium as 1:100, and IPTG were added at 0h respectively. The concentration of IPTG was 0, 10, 20, 50, 100, 200, 500, 1000 μM, and three parallel samples for each concentration. 200 μl of each sample were taken to measure the fluorescence after 6 hours of induction.
Results and Discussion
From the results shown in Fig.3，we can see that the improved T7-LacO promoter is much better than the control group in terms of lower basic leakage or higher expression status. At the zero point，that is without inducer, the leakage of improved promoter is more than ten times lower than the control group. While when enough inducer exist, that is, when the gene circuit is fully turned on, the maximum expression of the optimized promoter is not suppressed, and even slightly higher than the control group.
Furthermore, when comparing the switch ratios of the two inducible promoters, the ratio of the control group was only about 12, while the switch ratio of improved promoter could reach 147, which also shows that our modification of this part is effective.
Fig.3 Performance of improved promoter and original promoter
In the above experiments and results, we optimized the characterization performance of the T7-LacO promoter by adding an extra LacO site. This may be because in the lactose operon model, the LacI repressor is a tetramer that binds to the LacO site to control the state of inducible promoter. The function of LacO involves various factors such as DNA bending and interaction between protein and DNA. The specific influence mechanism of the amount of lacO on the promoter needs to be further studied.
