Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker

Heap, John T. and Ehsaan, Muhammad and Cooksley, Clare M. and Ng, Yen-Kuan and Cartman, Stephen T. and Winzer, Klaus and Minton, Nigel P. (2012) Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker. Nucleic Acids Research, 40 (8). 10/1-10/10. ISSN 0305-1048

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Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms.

Item Type: Article
Schools/Departments: University of Nottingham UK Campus > Faculty of Medicine and Health Sciences > School of Life Sciences > School of Molecular Medical Sciences
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Depositing User: Chamberlain, Mr Dick
Date Deposited: 29 Apr 2014 09:40
Last Modified: 13 Sep 2016 16:32

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