Project Background

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In order to study the expression and regulation of genes in cells directly, scholars put forward the concept of reporter, and develop many useful report systems. The luciferase lux， which found in the vibrio genome, is one of report systems. Bacterial luciferase is a flavin-dependent monooxygenase, catalyzing the reaction of O2 with reduced FMN and a long-chain aliphatic aldehyde, with concomitant emission of blue-green light. Compared with the popular reporter gene GFP, lux with hypo-toxicity and auto-luminance still worth further consideration and is of great potentiality. EvolvR, a system that can continuously diversify all nucleotides within a tunable window length at user-defined site, is an attractive method for continuous directed evolution. After the expression of user-designed gRNA, enCas9 is mediated to target and nick the user-defined loci on the genome or plasmid. The pol13M mutant with low fidelity degrades single strand from 5' to 3' from the nick and resynthesizes a new strand with a length of about 350bp. The EvolvR system enables targeted genes to mutate, express, be screened in vivo, thus, it achieves continuous directed evolution.

We believe that the EvolvR system is a capable continuous directed evolution tool, and the optimized luciferase system is worth expecting, so we set up the project.