Use of transposase and ends of IS608 enables precise and scarless genome modification for modulating gene expression and metabolic engineering applications in Escherichia coli

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dc.citation.journalTitleBiotechnology Journalen_US
dc.contributor.authorThakker, Chandreshen_US
dc.contributor.authorLin, Kevinen_US
dc.contributor.authorMartini-Stoica, Heidien_US
dc.contributor.authorBennett, George N.en_US
dc.date.accessioned2015-09-24T18:13:01Zen_US
dc.date.available2015-09-24T18:13:01Zen_US
dc.date.issued2015en_US
dc.description.abstractVarious methods have been developed for gene disruption in bacteria; however, extra in vitro manipulation steps or the residual presence of a scar in the host chromosome limits the use of such methods. By utilizing the unique properties of ISHp608, we have developed a simple and precise method for genome manipulation in Escherichia coli that alters the gene sequence without leaving foreign DNA in the chromosome. This strategy involves PCR amplification of a DNA cassette containing ISHp608-LE (left end)-antibiotic resistance gene-counterselection marker-ISHp608-RE (right end) by using primers containing extensions homologous to the adjacent regions of the target gene on the chromosome. The λ Red mediated recombination of the PCR product and antibiotic resistance screening results in transformants with a modified gene target. The ISHp608-LE-antibiotic resistance gene-counterselection marker-ISHp608-RE cassette can then be excised using a temperature sensitive plasmid expressing the TnpA transposase, which precisely cleaves ISHp608-LE and ISHp608-RE without leaving a scar sequence. We demonstrated lacZ gene point mutation repair, two precise disruptions of the lacZ gene and constructed a library of lacZ variants having variable β-galactosidase activity by changing its ribosome binding site sequences using the ISHp608 system. This technique can be used in E. coli genome modification and could be extended for use in other bacteria.en_US
dc.identifier.citationThakker, Chandresh, Lin, Kevin, Martini-Stoica, Heidi, et al.. "Use of transposase and ends of IS608 enables precise and scarless genome modification for modulating gene expression and metabolic engineering applications in Escherichia coli." <i>Biotechnology Journal,</i> (2015) Wiley: http://dx.doi.org/10.1002/biot.201500205.en_US
dc.identifier.doihttp://dx.doi.org/10.1002/biot.201500205en_US
dc.identifier.urihttps://hdl.handle.net/1911/81711en_US
dc.language.isoengen_US
dc.publisherWileyen_US
dc.rightsThis is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Wiley.en_US
dc.subject.keywordcounterselectionen_US
dc.subject.keywordgenome modificationen_US
dc.subject.keywordIS608en_US
dc.subject.keywordred recombinaseen_US
dc.subject.keywordtransposaseen_US
dc.titleUse of transposase and ends of IS608 enables precise and scarless genome modification for modulating gene expression and metabolic engineering applications in Escherichia colien_US
dc.typeJournal articleen_US
dc.type.dcmiTexten_US
dc.type.publicationpost-printen_US
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