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No review score entered. WHU-China 2023

EL222, a blue-light inducible promoter, is a typical single-component photosensitive system. pEL222 is a gene segment that can bind to EL222 protein. In darkness, the EL222 protein remains as a monomer without binding to pEL222 and inactivates the downstream gene. While under the blue light the two monomers are polymerized into a dimer and the dimer binds to pEL222, activating downstream genes. Results from previous teams have shown that two EL222 monomers can still bind together even in the darkness, causing downstream leakage. In our project, to reduce downstream leakage and lower the detection/sensitivity limit of EL222 —— our blue-light inducible promoter, we performed directed evolution on the binding region(pEL222) of EL222 by error-prone PCR. We introduced random mutations into pEL222 to alter the degree of the combination between pEL222 and EL222 proteins. eGFP is an enhanced green-fluorescent protein. It is a basic and wildly-used reporter protein. It can be activated by blue light and give out green light. In our experiments, eGFP is used as reporter in the first design. However, eGFP has an excitation peak at 488 nm, which is quite close to the 475nm we used to induce. sBFP2 is a basic blue fluorescent protein, it can be activated by UV light and emit blue light around 446nm. In our design, it is used as a reporter to replace eGFP. In this way, our observation and measurements will not be disturbed by quenching. mRFP1 is a basic red fluorescent protein, it can be activated by green light and emit red light around 607nm. Also, red fluorescence can be observed under UV light. In our design, it is used as a reporter to replace eGFP. In this way, our observation and measurements will not be disturbed by quenching.

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