A coelectroporation method for the isolation of cryptic plasmids from Lactococcus lactis


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Abstract

Aims:A coelectroporation method using a marker plasmid for indirect selection of lactococcal plasmids with unassigned functions was evaluated.Methods and Results:Cryptic plasmids were mixed with an erythromycin resistance (Eryr) marker plasmid and introduced into a recipient strain by electroporation, followed by plasmid extraction of erythromycin-resistant transformants. By optimizing the ratio between the marker plasmid and the cryptic plasmids, an average of 20% cotransformants was obtained, including combinations of more than one cryptic plasmid. The marker plasmid pSA3 was easily eliminated from the cotransformed cells by subculture without selective pressure.Conclusions:This cotransformation approach reduces the number of colonies that must be screened to find transformants harbouring cryptic plasmids.Significance and Impact of the Study:The method facilitates the isolation of cryptic plasmids, helps in assigning functions to unknown plasmids and allows construction of food-grade lactococcal strains with new combinations of wild-type plasmids.

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