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

Heap, John T.; Ehsaan, Muhammad; Cooksley, Clare M.; Ng, Yen-Kuan; Cartman, Stephen T.; Winzer, Klaus; Minton, Nigel P.

Authors

John T. Heap j.heap@imperial.ac.uk

Muhammad Ehsaan

Clare M. Cooksley

Yen-Kuan Ng

Stephen T. Cartman

Klaus Winzer klaus.winzer@nottingham.ac.uk

Nigel P. Minton nigel.minton@nottingham.ac.uk

Abstract

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.

Journal Article Type Article
Publication Date Jan 18, 2012
Journal Nucleic Acids Research
Print ISSN 0305-1048
Electronic ISSN 0305-1048
Publisher Oxford University Press (OUP)
Peer Reviewed Peer Reviewed
Volume 40
Issue 8
Article Number 10
Institution Citation Heap, J. T., Ehsaan, M., Cooksley, C. M., Ng, Y., Cartman, S. T., Winzer, K., & Minton, N. P. (2012). Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker. Nucleic Acids Research, 40(8), doi:10.1093/nar/gkr1321
DOI https://doi.org/10.1093/nar/gkr1321
Publisher URL http://nar.oxfordjournals.org/content/40/8/e59.full
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0




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