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dc.contributor.authorLeprince, Audrey
dc.contributor.authorde Lorenzo, Víctor
dc.contributor.authorVöller, Petra
dc.contributor.authorvan Passel, Mark W J
dc.contributor.authorMartins dos Santos, Vitor A P
dc.date.accessioned2012-08-27T12:34:58Z
dc.date.available2012-08-27T12:34:58Z
dc.date.issued2012-06
dc.identifier.citationRandom and cyclical deletion of large DNA segments in the genome of Pseudomonas putida. 2012, 14 (6):1444-53 Environ. Microbiol.en_GB
dc.identifier.issn1462-2920
dc.identifier.pmid22429517
dc.identifier.doi10.1111/j.1462-2920.2012.02730.x
dc.identifier.urihttp://hdl.handle.net/10033/240051
dc.description.abstractCumulative site-directed mutagenesis is of limited suitability for the global analysis of the gene functions in the microbe's cellular network. In order to simplify and stabilize the genome of the soil bacterium Pseudomonas putida, we developed a recyclable three-step excision method based on the combination of customized mini-transposons and the FLP-FRT site-specific recombination system. To demonstrate the powerful potential of these tools, we first established insertion mutant libraries that allow users to study gene functions with respect either to phenotypic characteristics (single insertions) or to their involvement in predicted networks (double insertions). Based on these libraries, we generated as a proof-of-principle, single-deletion mutants lacking ~4.1% of the genome (~3.7% of the gene repertoire). A cyclical application of the method generated four double-deletion mutants of which a maximum of ~7.4% of the chromosome (~6.9% of the gene count) was excised. This procedure demonstrates a new strategy for rapid genome streamlining and gain of new insights into the molecular interactions and regulations.
dc.language.isoenen
dc.rightsArchived with thanks to Environmental microbiologyen_GB
dc.titleRandom and cyclical deletion of large DNA segments in the genome of Pseudomonas putida.en
dc.typeArticleen
dc.contributor.departmentSystems and Synthetic Biology Group, Helmholtz-Centre for Infection Research, Braunschweig, Germany.en_GB
dc.identifier.journalEnvironmental microbiologyen_GB
refterms.dateFOA2013-06-15T00:00:00Z
html.description.abstractCumulative site-directed mutagenesis is of limited suitability for the global analysis of the gene functions in the microbe's cellular network. In order to simplify and stabilize the genome of the soil bacterium Pseudomonas putida, we developed a recyclable three-step excision method based on the combination of customized mini-transposons and the FLP-FRT site-specific recombination system. To demonstrate the powerful potential of these tools, we first established insertion mutant libraries that allow users to study gene functions with respect either to phenotypic characteristics (single insertions) or to their involvement in predicted networks (double insertions). Based on these libraries, we generated as a proof-of-principle, single-deletion mutants lacking ~4.1% of the genome (~3.7% of the gene repertoire). A cyclical application of the method generated four double-deletion mutants of which a maximum of ~7.4% of the chromosome (~6.9% of the gene count) was excised. This procedure demonstrates a new strategy for rapid genome streamlining and gain of new insights into the molecular interactions and regulations.


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