Team:Tsinghua-A/Contribution
Introduction
This project is intended to build blue light inducible expression system with high specificity and efficiency. This project is made of two composite parts, and the former one has 8 different design. The existing Part BBa_K2332001 shows previous work on blue light inducible expression system with GFP reporter (pBLind GFP). Based on this part, we wonder how the regulation factor photosensitive protein EL222 influence the light induction effect. Thus, we construct 8 different transcription conditions of EL222 in plasmid. After transformed, E.coli is cultured in blue light and dark environment for 12 hours. We measured the relative fluorescence intensity and calculated the radio of different environment. By analyzing the received data, we can finally get a shallow understanding of the specificity and efficiency.
figure 1: K2332001 Blue light inducible expression system. Under blue light, the EL222 DNA binding protein dimerises and bind its binding region within the designed Pblind promoter, overlapping the -35 region of the luxI promoter. This ultimately results in the recruitment of RNAP and transcriptional activation. Figure adapted from Jayaraman P. et al. (2016)
Source
The blue light inducible GFP expression system is the same as BBa_K2332001. The EL222 expression system is made of BBa_J23119 promoter family, BBa_B0034 RBS and BBa_B0015 terminator.
EXPERIMENT
In the part BBa_K2332001, reporter GFP is reporter of expression. By measuring relative fluorescence intensity, we can get Blue light induction effect. In the original design, this effect is not significant enough. It is determined by EL222 concentration, so we decided to change EL222 expression level to reach more obvious induction effect. As an improvement of BBa_K2332001, we test 4 different promoters and the presence or absence of terminator on EL222 expression. The following table shows how they are combined together.
The EL222 is constructed on high copy pSB4A5 and the photosensitive system is constructed on low copy pSB1K3. Parts J23100 through J23119 are a family of constitutive promoter parts isolated from a small combinatorial library. The selected promoters’ strength from strong to weak is: J23119, J23100, J23106, J23114. The presence of B0015 terminator is supposed to slight enhance expression. So, in figure 2, EL222 expression level is sequentially descending from left to right.
figure 2: BBa_K2332001 characterization. EL222 expression level is sequentially descending from left to right.
The result shows that stronger promoter of EL222 indeed increase the relative fluorescence intensity. However, the goal of our project is to increase the radio between blue light and dark environment. In this field, weaker promoter of EL222 shows a more obvious differences. A possible explanation is when EL222 is overexpression, its concentration is so high that EL222 can activate GFP expression in both blue light and dark situation. When EL222 is low expression, the difference of environment can works as the document of K2332001 says, caused the GFP expression difference between blue light and dark. The preliminary conclusion is using J23106 and B0015 to express EL222 can get significant light regulation of K2332001 system. More experiment and data is needed to improve this project.