Cloning of a copper resistance gene cluster from the cyanobacterium Synechocystis sp. PCC 6803 by recombineering recovery
Cloning of a copper resistance gene cluster from the cyanobacterium Synechocystis sp. PCC 6803 by recombineering recovery
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.
Cloning, Copper resistance gene cluster, Heterologous expression, Homologous recombination, Mutant complementation, Recombineering recovery
1872-1878
Gittins, John R.
c4d269cc-aae0-4182-bc81-78dc724f7d95
8 July 2015
Gittins, John R.
c4d269cc-aae0-4182-bc81-78dc724f7d95
Gittins, John R.
(2015)
Cloning of a copper resistance gene cluster from the cyanobacterium Synechocystis sp. PCC 6803 by recombineering recovery.
FEBS Letters, 589 (15), .
(doi:10.1016/j.febslet.2015.05.014).
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.
Text
Gittins_Cloning.pdf
- Accepted Manuscript
More information
Published date: 8 July 2015
Keywords:
Cloning, Copper resistance gene cluster, Heterologous expression, Homologous recombination, Mutant complementation, Recombineering recovery
Organisations:
Ocean and Earth Science
Identifiers
Local EPrints ID: 377098
URI: http://eprints.soton.ac.uk/id/eprint/377098
ISSN: 0014-5793
PURE UUID: 7b71ee3d-a801-40ea-b497-a1a04dca0974
Catalogue record
Date deposited: 15 May 2015 09:27
Last modified: 14 Mar 2024 19:56
Export record
Altmetrics
Download statistics
Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.
View more statistics