Electrotransformation of Lactobacillus casei L-49-4 using a shuttle vector pJLE121 constructed for Escherichia coll and lactobacilli

摘要

The shuttle vector pJLE121 (4691 bp) for Escherichia coli and lactobacilli was constructed. pJLE121 contained ori and repA of plasmid pLC494, which was isolated from Lactobacillus casei L-49, pMBl, ori of pJIR418, and the erythromycin resistance geneas the selection marker of Gram-positive and Gram-negative bacteria. The transformation efficiency of Lactobacillus casei L-49-4 (plasmid-free mutant of strain L-49) using pJLE121 was the highest when the cells (OD_(600) = 0.25) after cultivation in MRSbroth containing 1.0% glycine were washed with 10 mM MgCl_2 buffer and resuspended in 10% glycerol solution, and then electroporated at 1.75 kV, 200 0 and a capacitance of 25 mu F. This shuttle vector pJLE121 was transferred into some lactobacilli, and transformants obtained using pJLE121 were formed more faster than using shuttle vector (containing ori and repA of plasmid pLC494) containing chloramphenicol resistance gene as the selection marker of gram-positive.