Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Efficient and Precise Construction of Markerless Manipulations in the Bacillus subtilis Genome

  • Yu, Haojie (Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University) ;
  • Yan, Xin (Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University) ;
  • Shen, Weiliang (Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University) ;
  • Shen, Yujia (Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University) ;
  • Zhang, Ji (Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University) ;
  • Li, Shunpeng (Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University)
  • Published : 2010.01.31
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Abstract

We have developed an efficient and precise method for genome manipulations in Bacillus subtilis that allows rapid alteration of a gene sequence or multiple gene sequences without altering the chromosome in any other way. In our approach, the Escherichia coli toxin gene mazF, which was used as a counter-selectable marker, was placed under the control of a xylose-inducible expression system and associated with an antibiotic resistance gene to create a "mazF-cassette". A polymerase chain reaction (PCR)-generated fragment, consisting of two homology regions joined to the mazF-cassette, was integrated into the chromosome at the target locus by homologous recombination, using positive selection for antibiotic resistance. Then, the excision of the mazF-cassette from the chromosome by a single-crossover event between two short directly repeated (DR) sequences, included in the design of the PCR products, was achieved by counter-selection of mazF. We used this method efficiently and precisely to deliver a point mutation, to inactivate a specific gene, to delete a large genomic region, and to generate the in-frame deletion with minimal polar effects in the same background.

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References

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