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.

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.

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), Article 10. https://doi.org/10.1093/nar/gkr1321

• Journal Article Type: Article
• Publication Date: Jan 18, 2012
• Deposit Date: Apr 29, 2014
• Publicly Available Date: Apr 29, 2014
• Journal: Nucleic Acids Research
• Print ISSN: 0305-1048
• Electronic ISSN: 0305-1048
• Publisher: Oxford University Press
• Peer Reviewed: Peer Reviewed
• Volume: 40
• Issue: 8
• Article Number: 10
• DOI: https://doi.org/10.1093/nar/gkr1321
• Public URL: https://nottingham-repository.worktribe.com/output/709053
• Publisher URL: http://nar.oxfordjournals.org/content/40/8/e59.full