Abstract
Our Laboratory has isolated a new strain of Lactobacillus rhamnosus, named T CELL-1, from intestines of a healthy adult in Taiwan. Unfortunately, we were unable to detect any endogenous plasmid in it. For DNA manipulation, it is necessary to construct a plasmid which has a origin that allow it to replicate in Lb. rhamnosus T CELL-1.Therefore, we screened all other Lactobacilli in our Laboratory collection for native plasmid and tested whether they can replicate in Lb. rhamnosus T CELL-1.As a result, we found a plasmid, pLP1, being suitable as a cloning vector, from one of the 13 strains. The plasmid was amplified pLP1 by inverse PCR and constructed into a shuttle vector, pBLP, which could replicate in both E. coli and Lactobacilli. An antibiotic-resistance gene, ermC, was inserted as a selection marker and the resulting plasmid was named pBLPE. The recombinant plasmid was subsequently transformed into Lb. rhamnosus T CELL-1 by eletroporation. The best condition for eletroporation mediated transformation of this Lactobacillus was also determined. Plasmid pBLPE is somewhat unstable in Lb. rhamnosus T CELL-1 and no complete plasmid was detected. To solve the problem of plasmid instability, we deleted the region derived from E. coli and self- ligated remaining portion to produce a new recombinant DNA, pLPE. The latter, pLPE, was more stable than the former, pBLPE. The experiment revealed that it had a problem of structure after eletrotransformation and the sequence would be changed, with Southern hybridization and several primers of PCR. Nevertheless, some minor sequence alterations of the plasmid also detected after it was introduced into Lb. rhamnosus TCELL-1.The details of this sequence change remain to be identified.