Cloning of lactobacillus casei allosteric lactate dehydrogenase gene in escherichia coli

Abstract

An attempt was made to clone an allosteric lactate dehydrogenase (LDH) gene of Lactobacillus casei in Escherichia coli. The chromosomal DNA of L. casei was restricted and hybridized with a synthetic probe. Plasmid pUC9 was used as a vector. A mixture of oligonucleotides was synthesized by modified phosphite triester methdo based on the nucleotide sequence of the LDH produced by L. casei. The synthesized probe was composed of 17 bases and was labeled with [$\gamma-^{32}P$] ATP before use. The chromosomal DNA of L. casei was digested with StuI and then hybridized with the labeled probe following the procedures of Southern hybridization. The probe hybridized to a 2.3 killobase (kb) chromosomal DNA fragment. Again the chromosomal DNA of L. casei was digested with StuI and DNA fragments of 2.3 kb size were isolated. The isolated DNA fragments were ligated to SmaI site of pUC9. With the ligation mixture E. coli JM83 was transformed to make a gene library. Approximately 1500 E. coli transformants were toothpicked and subjected to in situ colony hybridization. Thirty two clones out of the 1500 colonies showed positive hybridization signals. Plasmids were isolated from eight clones selected randomly out of the 32 clones. Restriction analysis proved all of the isolated plasmids were identical harboring the 2.3 kb chromosomal fragment of L. casei. The E. coli transformants harboring the recombinant plasmids, however, did not show LDH activity. This may due to 1) damage of LDH gene by StuI, 2) the inactive promoter of LDH gene of L. casei in E. coli, or to 3) a wrong probe. To elucidate these possibilities, the nucleotide sequences of the inserted L. casei chromosomal fragments were analyzed. The nucleotide sequence analysis revealed that all of the 17 bases of the probe matched well with the bases of the chromosomal DNA except one base "T" mismatched with "C". This C-T mismatch might have picked chromosomal DNA fragments other than the LDH gene. Since the probe was constr...