Homology-dependent recombination of large synthetic pathways into E. coli genome via λ-Red and CRISPR/Cas9 dependent selection methodology

摘要

Outline of the λ-Red assisted homology-dependent recombination for large synthetic pathways integration in . Construction of plasmid pRC-IS5 with large synthetic pathways. pRC-IS5 (including R6K and a homologous region) replicates normally in with the expression of pir + protein and the plasmid replication was restricted in normal . Single-crossover HDR assisted by λRed. The vector pRC-IS5 was introduced into the host which harbored pCas with the expression of Exo, Beta, and Gam, and then selection was conducted with the addition of chloramphenicol. Single crossover produced homology-dependent insertion events, where the entire vector pRC-IS5 was integrated into the chromosome at the target locus. A simple screening step by PCR diagnosis could identify the desired mutant. Deleting redundant sequences assisted by λ-Red. The gRNA plasmid pTargetF-delete and the donor template were electroporated into the competent cells harbored plasmid pCas with the expression of Cas9 nuclease and λ-Red protein, and then the selection was carried out using kanamycin and spectinomycin. λ-Red mediated deletion at the lagging strand of the replication fork produced homologous recombination, where the redundant sequences were deleted