Blue Light-inducible Semi-random Mutagenesis on Specific Exogenous DNA in Escherichia coli BL21 Strain

Mutagenesis technologies have been widely used in variation breeding, directed evolution, and protein engineering. However, most methods can only induce random genomic DNA mutations that are uncontrollable, leading to deformities or even lethal effects. Here, by leveraging the fusion of three systems: cytosine base editor (CBE), blue light-inducible p-Mag/n-Mag elements, and split T7 RNA polymerase, this outlined method incorporates a blue-light-controllable, DNA region-specific, semi-random mutagenesis system. This system can mutate cytosine to thymine on DNAs starting from the T7 promoter and ending in the T7 terminator. Any exogenous gene downstream of the T7 promoter can be potentially mutated. These tools were also designed in two parts to make the system blue-light-controllable. The first part was created by fusing CBE with n-Mag and the N-terminal-T7 RNA polymerase gene. The second part involved fusing p-Mag with the C-terminal-T7 RNA polymerase. Under blue light, these two parts were assembled to function as a mutation generator on DNAs between the T7 promoter and the T7 terminator. Without blue light, p-Mag is detached from n-Mag, releasing the CBE from editing the DNA. The aim of this protocol is to generate region-specific random mutations in Escherichia coli with blue light control and further validate the mutated plasmids. The system can be adapted to an in vivo evolution system where the function of the gene of interest can be selected, and the desired mutations can be collected for further study.