Fig. 1

The optimization expression strategies for T7 RNAP and pET plasmids. A Illustration of protein expression of recombinant protein genes on pET plasmids. B Optimization of T7 RNAP transcription and translation level, including substitutions of different promoters, and mutations in promoter functional region and RBS sequence. C regulation of T7 RNAP activity. The conventional approach is to utilize lysozyme or light-induction to regulate. D Optimization of pET plasmids based on expression intensity and copy numbers. Among them, the expression intensity was optimized by constructing an ITR library to screen for optimal expression results. The degree of binding of RNA-i to RNA-p determines the replication intensity of the plasmid to control the copy numbers. By constructing a promoter library for RNA-p, replacing the inducible promoter, and using dCas9 to regulate expression intensity, the copy numbers can be controlled