pBR322 vector

Full-length RecE and RecT from the Rac prophage mediate efficient linear-linear homologous recombination and can be used to clone large DNA regions directly from genomic DNA into expression vectors, bypassing library construction and screening. Homologous recombination mediated by the lambda phage Redαβ has been widely used in recombinant DNA engineering. In this study, researchers present a protocol for the direct cloning and engineering of biosynthetic gene clusters, large operons, or individual genes from genomic DNA using an E. coli host with the RecET and Redαβ systems. The pipeline uses standardized cassettes for horizontal gene transfer options, as well as vectors with different origins of replication, configured to minimize recombination background by using selective replication templates or CcdB counterselection. These optimized reagents and protocols facilitate rapid acquisition of transgenes from genomic DNA preparations and are ready for heterologous expression within 1 week.

In this study, the strategy for heterologous expression was based on two recombineering systems in one E. coli host. The plasmid-produced RecET protein is expressed from an L-arabinose-responsive BAD promoter. The Redαβ gene is integrated into the chromosome and expressed from the L-rhamnose-responsive rhaSR promoter. Digested genomic DNA carrying the gene cluster of interest and a linear cloning vector were co-electroporated into arabinose-induced RecET-expressing E. coli cells for direct cloning of the gene cluster by LLHR. The clones obtained can be further modified in several different ways by Redαβ recombineering, including: (i) the pBR322 and the p15A vectors can be exchanged with the broad-hostrange replicon of Bordetella bronchiseptica pBBR1 plasmid for replication in Gramnegative bacteria, and (ii) the conjugation and transposable cassette (oriT-tnpA-IR) or the conjugation and site-specific integration cassette (oriT-attP-phiC31) can be inserted into the three vectors using standardized homology arms. The final construct is transferred into a tractable host for heterologous expression.

Figure 1. Strategy diagram for direct cloning and engineering of natural product biosynthetic gene clusters for heterologous expression. (Wang, Hailong, et al. 2016)