An improved method for oriT-directed cloning and functionalization of large bacterial genomic regions.

ABSTRACT: We have made significant improvements to a broad-host-range system for the cloning and manipulation of large bacterial genomic regions based on site-specific recombination between directly repeated oriT sites during conjugation. Using two suicide capture vectors carrying flanking homology regions, oriT sites are recombined on either side of the target region. Using a broad-host-range conjugation helper plasmid, the region between the oriT sites is conjugated into an Escherichia coli recipient strain, where it is circularized and maintained as a chimeric mini-F vector. The cloned target region is functionalized in multiple ways to accommodate downstream manipulation. The target region is flanked with Gateway attB sites for recombination into other vectors and by rare 18-bp I-SceI restrictio

SUBMITTER: Kvitko BH 

PROVIDER: S-EPMC3754689 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature