The University of Southampton
University of Southampton Institutional Repository

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
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
0014-5793
1872-1878
Gittins, John R.
c4d269cc-aae0-4182-bc81-78dc724f7d95
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), 1872-1878. (doi:10.1016/j.febslet.2015.05.014).

Record type: Article

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
Download (778kB)

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: 26 Apr 2022 17:23

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

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of http://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×