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Cloning of a copper resistance gene cluster from the cyanobacterium Synechocystis sp. PCC 6803 by recombineering recovery
Author(s) -
Gittins John R.
Publication year - 2015
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2015.05.014
Subject(s) - recombineering , plasmid , complementation , gene , gene cluster , synechocystis , homologous recombination , biology , cloning (programming) , escherichia coli , dna , genetics , mutant , computer science , programming language
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.