An Improved Method for oriT -Directed Cloning and Functionalization of Large Bacterial Genomic Regions

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 repeatedoriT 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 theoriT sites is conjugated into anEscherichia 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 GatewayattB sites for recombination into other vectors and by rare 18-bp I-SceI restriction sites for subcloning. The Tn7 -functionalized target can also be inserted at a naturally occurring chromosomalatt Tn7 site(s) or maintained as a broad-host-range plasmid for complementation or heterologous expression studies. We have used the oriTn7 capture technique to clone and complementBurkholderia pseudomallei genomic regions up to 140 kb in size and have created isogenicBurkholderia strains with various combinations of genomic islands. We believe this system will greatly aid the cloning and genetic analysis of genomic islands, biosynthetic gene clusters, and large open reading frames.