Construction of a Lactococcal Shuttle/Expression Vector Containing a $\beta$-Galactosidase Gene as a Screening Marker

선별마커로써 $\beta$-Galactosidase 유전자를 포함한 Lactococcus용 셔틀/발현 벡터 제조

  • Han Tae Un (School of Agricultural Biotechnology, Seoul National University) ;
  • Jeong Do-Won (School of Agricultural Biotechnology, Seoul National University) ;
  • Cho San Ho (School of Agricultural Biotechnology, Seoul National University) ;
  • Lee Jong-Hoon (Department of Food Science and Biotechnology, Kyonggi University) ;
  • Chung Dae Kyun (Department of Genetic Engineering, KyungHee University) ;
  • Lee Hyong Joo (School of Agricultural Biotechnology, Seoul National University)
  • 한태운 (서울대학교 농생명공학부) ;
  • 정도원 (서울대학교 농생명공학부) ;
  • 조산호 (서울대학교 농생명공학부) ;
  • 이종훈 (경기대학교 식품생물공학과) ;
  • 정대균 (경희대학교 유전공학과) ;
  • 이형주 (서울대학교 농생명공학부)
  • Published : 2005.12.01

Abstract

A new lactococcal shuttle/expression vector for lactococci, pWgal13T, was constructed using a $\beta$-galactosi-dase gene (lacZ) from Lacfococcus lactis ssp. lactis ATCC 7962 as a screening marker. The pWgal 13T was introduced into Escherichia coli DH5a and L. lactis MG1363, and was easily detected by the formation of blue colonies on a medium containing X-gal without any false transformants. Also, the quantitatively lacZ activity of pWgal13T was measured in L. lactis ssp. cremoris MG1363, and was found to be four times higher than that of L. lactis ssp. lactis ATCC7962 grown on a medium containing glucose, which shows that the lacZ gene of pWgal13T can be used for the efficient screening of L. lactis on general media. The pWgal13T was equipped with a lactococcal replicon of pWV01 from L. lactis Wg2, the new promoter P13C from L. lactis ssp. cremoris LM0230, multiple cloning sites, and a terminator for the expression of a relevant gene. The vee-tor pWgal13T was used for the expression of the EGFP gene in E. coli and L. lactis. These results show that the lactococcal expression/shuttle vector constructed in the present study can be used for the production of foreign proteins in E. coli and L. lactis.

Keywords

Lactococcal shuttle/expression vector;Lactococcus lactis;lacZ;P13C;EGFP protein

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