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

Title: Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker.
Author(s): Heap, JT
Ehsaan, M
Cooksley, CM
Ng, YK
Cartman, ST
Winzer, K
Minton, NP
Item Type: Journal Article
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. © 2011 The Author(s).
Publication Date: 1-Apr-2012
Start Page: e59
Journal / Book Title: Nucleic Acids Res
Volume: 40
Issue: 8
Copyright Statement: © The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Conference Place: England
Appears in Collections:Faculty of Natural Sciences

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