High efficiency electrotransformation of Lactococcus lactis spp. lactis cells pretreated with lithium acetate and dithiothreitol

Electrotransformation efficiencies of up to 105 transformants per μg DNA have been reported in the literature for L. lactis spp.lactis LM0230. We report here that treatment with LiAc and DDT before electroporation increased transformation efficiency to 225 ± 52.5 × 107 transformants per μg DNA, while with untreated cells or treated with LiAc alone transformation efficiency approximated 1.2 ± 0.5 × 105 transformants per μg DNA. Results of the same trend were obtained with L. lactis ATCC 11454, although transformation efficiency of this strain was significantly lower. No difference was found in the survival rate of pretreated cells after electroporation. Transformation efficiency was found to vary directly with cell density and that of 1010 cells/ml resulted in the highest efficiencies. Following electrotransformation of pretreated cells with LiAc and DDT, pTRKH3 stability was examined. Both host-vector systems proved to be reproducible and highly efficient.This investigation sought to improve still further transformation efficiencies and to provide a reliable high efficiency transformation system for L. lactis spp. lactis. The applied methodology, tested in two well-known strains, allows the production of large numbers of transformants and the construction of large recombinant libraries.Lactococcus lactis is the model lactic acid bacterium extensively used in the manufacture of fermented foods of animal origin. A goal for the food industry has always been to improve L. lactis strains and to stabilize important traits of this bacterium. Today, genetic engineering is used extensively for manipulating L. lactis. Important contributions to the evolution of genetic technology of this organism include the development of transformation techniques and the construction of powerful plasmids for gene cloning and for general mutagenesis [1,2]. In the past years, electroporation has become the widest used method for introducing DNA in L. lactis cells. Transformation by electropor