The ClosTron: Mutagenesis in Clostridium refined and streamlined
The recent development of the ClosTron Group II intron directed mutagenesis tool for Clostridium has advanced genetics in this genus, and here we present several significant improvements. We have shown how marker re-cycling can be used to construct strains with multiple mutations, demonstrated using...
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Published in | Journal of microbiological methods Vol. 80; no. 1; pp. 49 - 55 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
Amsterdam
Elsevier B.V
2010
Elsevier |
Subjects | |
Online Access | Get full text |
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Summary: | The recent development of the ClosTron Group II intron directed mutagenesis tool for
Clostridium has advanced genetics in this genus, and here we present several significant improvements. We have shown how marker re-cycling can be used to construct strains with multiple mutations, demonstrated using FLP/FRT in
Clostridium acetobutylicum; tested the capacity of the system for the delivery of transgenes to the chromosome of
Clostridium sporogenes, which proved feasible for 1.0
kbp transgenes in addition to a marker; and extended the host range of the system, constructing mutants in
Clostridium beijerinckii and, for the first time, in a B1/NAP1/027 ‘epidemic’ strain of
Clostridium difficile. Automated intron design bioinformatics are now available free-of-charge at our website
http://clostron.com; the out-sourced construction of re-targeted intron plasmids has become cost-effective as well as rapid; and the combination of constitutive intron expression with direct selection for intron insertions has made mutant isolation trivial. These developments mean mutants can now be constructed with very little time and effort for the researcher. Those who prefer to construct plasmids in-house are no longer reliant on a commercial kit, as a mixture of two new plasmids provides unlimited template for intron re-targeting by Splicing by Overlap Extension (SOE) PCR. The new ClosTron plasmids also offer blue–white screening and other options for identification of recombinant plasmids. The improved ClosTron system supersedes the prototype plasmid pMTL007 and the original method, and exploits the potential of Group II introns more fully. |
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ISSN: | 0167-7012 1872-8359 |
DOI: | 10.1016/j.mimet.2009.10.018 |