title: Cloning of a copper resistance gene cluster from the cyanobacterium Synechocystis sp. PCC 6803 by recombineering recovery.
abstract: A copper resistance gene cluster (6 genes, ∼8.2 kb) was isolated from the cyanobacterium Synechocystis sp. PCC 6803 by recombineering recovery (RR). Following integration of a narrow-host-range plasmid vector adjacent to the target region in the Synechocystis genome (pSYSX), DNA was isolated from transformed cells and the plasmid plus flanking sequence circularized by recombineering to precisely clone the gene cluster. Complementation of a copper-sensitive Escherichia coli mutant demonstrated the functionality of the pcopM gene encoding a copper-binding protein. RR provides a novel alternative method for cloning large DNA fragments from species that can be transformed by homologous recombination.
authors.affiliation: Ocean and Earth Science, University of Southampton, Waterfront Campus, National Oceanography Centre, Southampton SO14 3ZH, UK. Electronic address: jrgi@noc.soton.ac.uk.
authors.forename: John R
authors.initials: JR
authors.lastname: Gittins
mesh_terms: D003300:Copper / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D005810:Multigene Family; D009154:Mutation; D010957:Plasmids; D046939:Synechocystis / Q000235:genetics*
chemical_list: D003300:Copper
keywords: Cloning; Copper resistance gene cluster; Heterologous expression; Homologous recombination; Mutant complementation; Recombineering recovery