Our improvement project aims to address the high leakage problem in the blue light-inducible promoter system based on the natural photosensitive DNA-binding protein EL222. Previous efforts (XMU-China 2021) partially mitigated the leakage issue by replacing the strong promoter BBa_J23106 with pBlind. However, the system still exhibited notable gene expression in the absence of blue light.

To further reduce system leakage, we made several improvements. We inserted the RAT terminator sequence (BBa_K5398612) between the pBlind promoter and sfGFP reporter gene to prevent premature expression. Additionally, we co-expressed LicV (BBa_K5398611), a fusion protein with the RNA-binding domain of LicT and Vivid (VVD), driven by the strong promoter J23104, to stabilize the RAT element under blue light. We constructed pCDFDuet-1-EL222-RAT (BBa_K5398630) and pQE-60-LicV (BBa_K5398631) plasmids and transformed them into BL21(DE3) to achieve the desired functionality.

Our modifications successfully reduced the leakage level in the absence of blue light, significantly lowering background expression. The RAT terminator effectively prevented gene expression without blue light, while the combination of RAT and LicV regulated expression under blue light conditions, providing a more robust and tunable blue light-inducible promoter system.

Table 1 | Improved Part