Construction of recombinant Lactobacillus casei strains using splicing by overlap extension.
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.
Jeong
Do-Won
DW
Department of Agricultural Biotechnology, and 2Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea.
Lee
Jong-Hoon
JH
Lee
Hyong Joo
HJ
D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007780:Lacticaseibacillus casei / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D004269:DNA, Bacterial; D004274:DNA, Recombinant; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins