Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Construction and Transformation of an Endogenous Plasmid pBL1-free Brevibacterium lactofermentum

내재형 Plasmid pBL1이 제거된 Brevibacterium lactofermentum 개발과 형질전환

  • 이규남 (한국과학기술연구원 생명공학연구소) ;
  • 민본홍 (건국대학교 생화학과) ;
  • 윤기홍 (한국과학기술연구원 생명공학연구소)
  • Published : 1995.05.01
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Abstract

An endogenous cryptic plasmid, pBL1, which has been used to construct plasmid vectors for coryneform bacteria producing amino acids, was eliminated from Brevibacterium lactofermentum. The pBL1 was partially digested with Sau3AI and the resulting DNA fragments were subcloned into a suicide vector pEM1 which contains a kanamycin-resistant (km$^{r}$) gene. KM$^{r}$ B. lactofermentum transconjugants were obtained by conjugal transfer of the pEM1 derivatives containing pBL1 DNA fragments from Escherichia coli into B. lactofermentum. A km$^{r}$ transconjugant was analyzed to contain a plasmid pEB14, which occurred in vivo by homologous recombination between pBL1 and the conjugal-transferred plasmid. The pEB14 including the pEM1-derived km$^{r}$ gene was found to be lost concomitantly with km$^{r}$ phenotype, resulting in the construction of a pBL1-free strain of B lactofermentum. Based on transformation efficiencies and plasmid stability, the resultant pBL1- free strain is more useful than wild strain as a host cell for genetic manipulation. It could be concluded that foreign plasmid DNAs are efficiently isolated and analyzed from the pBL1-free strain because of the absence of endogenous pBL1 plasmid.

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References

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