N Corneau1, C Dubé, G LaPointe, E Emond . 1. Département de biochimie, Faculté des sciences et génie, Université Laval, Québec, Canada.
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. CONCLUSION: 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.
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. CONCLUSION: 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.