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Bacillus subtilis genome editing using ssDNA with short homology regions

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dc.creator Wang, Yang
dc.creator Weng, Jun
dc.creator Waseem, Raza
dc.creator Yin, Xihou
dc.creator Zhang, Ruifu
dc.creator Shen, Qirong
dc.date.accessioned 2012-08-22T14:56:52Z
dc.date.available 2012-08-22T14:56:52Z
dc.date.issued 2012-03-15
dc.identifier.citation Wang, Y., Weng, J., Waseem, R., Yin, X., Zhang, R., & Shen, Q. (2012). Bacillus subtilis genome editing using ssDNA with short homology regions. Nucleic Acids Research, 40(12), e91. doi: 10.1093/nar/gks248 en_US
dc.identifier.uri http://hdl.handle.net/1957/32738
dc.description This is the publisher’s final pdf. The published article is copyrighted by Oxford University Press and can be found at: http://nar.oxfordjournals.org/. en_US
dc.description.abstract In this study, we developed a simple and efficient Bacillus subtilis genome editing method in which targeted gene(s) could be inactivated by single-stranded PCR product(s) flanked by short homology regions and in-frame deletion could be achieved by incubating the transformants at 42 degrees C. In this process, homologous recombination (HR) was promoted by the lambda beta protein synthesized under the control of promoter P[subscript RM] in the lambda cI857 P[subscript RM]-P[subscript R] promoter system on a temperature sensitive plasmid pWY121. Promoter P[subscript R] drove the expression of the recombinase gene cre at 42 degrees C for excising the floxed (lox sites flanked) disruption cassette that contained a bleomycin resistance marker and a heat inducible counter-selectable marker (hewl, encoding hen egg white lysozyme). Then, we amplified the single-stranded disruption cassette using the primers that carried 70 nt homology extensions corresponding to the regions flanking the target gene. By transforming the respective PCR products into the B. subtilis that harbored pWY121 and incubating the resultant mutants at 42 degrees C, we knocked out multiple genes in the same genetic background with no marker left. This process is simple and efficient and can be widely applied to large-scale genome analysis of recalcitrant Bacillus species. en_US
dc.description.sponsorship Funding for open access charge: Priority Academic Program Development of Jiangsu Higher Education Institutions; Chinese Ministry of Science and Technology (2011BAD11B03-02) and Chinese Ministry of Agriculture (201103004). en_US
dc.language.iso en_US en_US
dc.publisher Oxford University Press en_US
dc.relation.ispartofseries Nucleic Acids Research en_US
dc.relation.ispartofseries Vol. 40 no. 12 en_US
dc.subject Gram positive bacteria en_US
dc.subject Single stranded DNA en_US
dc.subject Escherichia coli en_US
dc.subject Selectable marker en_US
dc.subject PRC products en_US
dc.subject Recombination en_US
dc.subject Lysozyme en_US
dc.subject Sequence en_US
dc.subject Transformation en_US
dc.subject system en_US
dc.title Bacillus subtilis genome editing using ssDNA with short homology regions en_US
dc.type Article en_US
dc.description.peerreview yes en_US
dc.identifier.doi 10.1093/nar/gks248

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