Cloning of circular DNAs from microorganisms using a novel plasmid capture system.

Plasmid capture system (PCS) facilitates cloning and manipulation of circular double-stranded DNA. We recently developed an improved PCS (PCS-LZ) to clone relatively large DNA molecules of 30-150 kb. The PCS-LZ donor consists of a mini-F replicon and a kanamycin resistance marker between Tn7 left and Tn7 right ends. Both the replicon and marker gene of the PCS-LZ donor are transferred into target plasmid DNAs by in vitro transposition, followed by replication in E. coli. Colonies are tested for lacZ expression by blue/white screening. Circular DNAs were obtained from plasmids of Bacillus thuringiensis, genome segments of Cotesia glomerata bracovirus and polymorphic genomes of Autographa californica nucleopolyhedrovirus. PCS-LZ is a powerful tool for use in genomic analysis and mutagenesis in microorganisms including invertebrate pathogens.