Cloning of a copper resistance gene cluster from the cyanobacterium Synechocystis sp. PCC 6803 by recombineering recovery

• Published in: FEBS letters Vol. 589; no. 15; pp. 1872 - 1878
• Main Author: Gittins, John R.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 08-07-2015
• Subjects: Cloning; Copper - pharmacology; Copper resistance gene cluster; Cyanobacteria; Drug Resistance, Bacterial - genetics; Escherichia coli; Escherichia coli - genetics; Genes, Bacterial; Genetic Complementation Test; Genetic Vectors; Heterologous expression; Homologous recombination; Multigene Family; Mutant complementation; Mutation; Plasmids; Recombineering recovery; Synechocystis; Synechocystis - genetics; Copper resistance gene cluster; Mutant complementation; Cloning; Homologous recombination; Recombineering recovery; Heterologous expression
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2015.05.014

•A Synechocystis copper resistance gene cluster was cloned by recombineering recovery (RR).•Functionality of the pcopM gene in E. coli was confirmed by mutant complementation.•RR represents an efficient way of isolating large DNA fragments.•The method should be applicable to species transformed by homologous recombination.
A copper resistance gene cluster (6 genes, ∼8.2kb) 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.