Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Construction of Recombinant Lactobacillus casei Strains Using Splicing by Overlap Extension

  • Jeong, Do-Won (Department of Agricultural Biotechnology, Seoul National University) ;
  • Lee, Jong-Hoon (Department of Food Science and Biotechnology, Kyonggi University) ;
  • Lee, Hyong-Joo (Department of Agricultural Biotechnology, Seoul National University)
  • Published : 2008.12.31
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Abstract

Recombinant Lactobacillus strains have been constructed using gene splicing by overlap extension (SOE). Primers were designed of which one end of an amplified product contained complementary sequences for an end of other amplified fragment. For efficient matching, we used an asymmetric PCR step that was effective at generating an excess of strands that would anneal in the final PCR. CP12, a recombinant fragment consisting of the integrase gene and attachment site of the bacteriophage A2, was constructed and inserted into the genome of Lactobacillus casei ATCC 393, yielding Lb. casei ATCC 393::XCP12. Another recombinant Lb. casei strain was constructed, where the egfp gene was a part of the construction. The EGFP produced from Lb. casei ATCC 393::XCEGFP14 was detected by Western blot hybridization. This simple and widely applicable approach has significant advantages over standard recombinant DNA techniques for Lactobacillus species.

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References

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