A single vector-based strategy for marker-less gene replacement in Synechocystis sp. PCC 6803

Microbial Cell Factories

Research

Dario Leister¹  Thilo Rühle¹  Stefania Viola¹ 

[1] Department Biology I, Ludwig-Maximilians-Universität München, Großhaderner Str. 2, D-82152, Planegg, Martinsried, Germany;

关键词: Genetic engineering;    Gene replacement;    Homologous recombination;    Synechocystis;   

DOI  :  10.1186/1475-2859-13-4

 received in 2013-11-04, accepted in 2014-01-03,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundThe cyanobacterium Synechocystis sp. PCC 6803 is widely used for research on photosynthesis and circadian rhythms, and also finds application in sustainable biotechnologies. Synechocystis is naturally transformable and undergoes homologous recombination, which enables the development of a variety of tools for genetic and genomic manipulations. To generate multiple gene deletions and/or replacements, marker-less manipulation methods based on counter-selection are generally employed. Currently available methods require two transformation steps with different DNA plasmids.ResultsIn this study, we present a marker-less gene deletion and replacement strategy in Synechocystis sp. PCC 6803 which needs only a single transformation step. The method utilizes an nptI-sacB double selection cassette and exploits the ability of the cyanobacterium to undergo two successive genomic recombination events via double and single crossing-over upon application of appropriate selective procedures.ConclusionsBy reducing the number of cloning steps, this strategy will facilitate gene manipulation, gain-of-function studies, and automated screening of mutants.

【 授权许可】

Unknown   
© Viola et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.