Team:XJTU-CHINA/Improve
Improvement of BBa_K2598038
Overview
This year team XJTU-CHINA aim to improve a well-characterized red-light-activated sensor which is part of the GRB system of 2018 team UCAS-China(Part:BBa_K2598038). This part contains red-light sensor cph8(BBa_I15010) and repressor CI (BBa_C0051) under its regulation. Base on this red-light-activated sensor, we add a CI regulated promoter - Pλ to this part and enable it to realize the function of two-phases switch (BBa_K3052030). Besides, we also added two fluorescent proteins - superfolder GFP (BBa_K3052041) and mRFP (BBa_K3052042) under the control of pomp (BBa_R0082) and Pλ(BBa_K1145005) respectively to report the expression level of each phase. Theoretically, RFP will expresses under red light and GFP expresses in the absence of red light.
UCAS-2018 tested the change of fluorescence intensity with the change of illumination intensity. When there is no light, fluorescence is not expressed, but when there is light, fluorescence starts to be expressed, and with the increase of time and the increase of light intensity, fluorescence intensity increases, which is shown as a one-way switch.
Cite:https://2018.igem.org/Team:UCAS-China/Demonstrate
Characterization
The light-fluorescent circuits (see circuits) were constructed by two-step Golden Gate Assembly and were confirmed by colony PCR. The length of this fragment is 1467 bp and the following results showed the successful constructions.
In order to validate the effectiveness of this switch, we qualitatively measure the fluorescent of this circuit at different time. Sample of DH5α E. coli transformed with BBa_K3052030 is incubated for 24 hours without red light and then continue incubating 24 hours with red light. We photographed both the sample of control group (DH5α E.coli wildtype) and experimental group(E.coli with light-fluorescent circuit ) by a fluorescence microscope at 24 hours (24 hours without light treatment) and 48 hours (24 h without light treatment and 24 h with red light treatment). The figure indicated that the bidirectional switch was functional to switch from GFP to RFP.
Furthermore, we used COFOCAL to sensitively detect GFP and RFP while switch is shifting. After the sample above had been incubated for 24 hours without light treatment, it was treated with intense red light (wavelength=640nm). After incubating for another 3 hours with red light treatment, we took out some of the sample and perform CONFOCAL imaging. The result shows that the RFP was appearing with red light treatment, which indicates this switch can be effectively triggered by red light.
To quantitatively test the efficiency of this light switch, we decided to cultivate the cells in dark environment first and then trigger the switch with intense red light. When they had been incubated for 30 hours without red light, we start to put intense red light around the medium. During the experiment, we measured the GFP and RFP intensity of the sample every 2 hours.
From Figure 4 we can see that the GFP intensity dropped sharply to a low and stable value in 4 hours when the sample was treated with red light, additionally the increase of RFP intensity also indicates the effectiveness of this bidirection switch.