NEW PROTOCOL FOR TRANSFORMING LACTOBACILLUS CASEI USING GROWTH IN HIGH SALT

MARÍA MERCEDES PALOMINO; MARIANO PRADO ACOSTA; MARIANA CLAUDIA ALLIEVI; CARMEN SÁNCHEZ RIVAS; SANDRA M RUZAL

San Miguel de Tucumán Argentina

Congreso; XLV SAIB. 2009; 2009

SAIB

Resumen: Because of their widespread industrial and medical importance, there is considerable interest in the manipulation and improvement of Lactobacillus strains using modern genetic engineering techniques.Limited transformability is in part consequence of their cell wall composition that is highly resistant to disruption by mechanical or cell-wall hydrolases treatments.We have described that L. casei BL23 grown in high salt shows modifications of their cell-wall that increases their sensitivity to lysis (Piuri et al. 2005).Here we report a new method based on the cell wall weakening by incorporation of high salt into the culture medium that enhanced the transformation frequency. For this purpose, L. casei was grown overnight in media containing 0.9 M of NaCl, carefully washed and kept at -70¨¬C bef Because of their widespread industrial and medical importance, there is considerable interest in the manipulation and improvement of Lactobacillus strains using modern genetic engineering techniques.Limited transformability is in part consequence of their cell wall composition that is highly resistant to disruption by mechanical or cell-wall hydrolases treatments.We have described that L. casei BL23 grown in high salt shows modifications of their cell-wall that increases their sensitivity to lysis (Piuri et al. 2005).Here we report a new method based on the cell wall weakening by incorporation of high salt into the culture medium that enhanced the transformation frequency. For this purpose, L. casei was grown overnight in media containing 0.9 M of NaCl, carefully washed and kept at -70¨¬C before submitting to different electroporation conditions. Consistent frequencies of >105 transformants per ¥ìg plasmid DNA have been obtained using an electroporation voltage of 12.5 kV cm-1. Various plasmids have been assayed and been successfully transferred: pNZ235, pGK13, pIL253, pRV610, pRV620.Comparison of this method to others using lysozyme, glycine or penicillin as cell wall weakening agents are discussed.At the moment we are trying to extend this novel method to other less transformable Lactobacillus strains such as L. plantarum, sakei and curvatus. ore submitting to different electroporation conditions. Consistent frequencies of >105 transformants per ¥ìg plasmid DNA have been obtained using an electroporation voltage of 12.5 kV cm-1. Various plasmids have been assayed and been successfully transferred: pNZ235, pGK13, pIL253, pRV610, pRV620.Comparison of this method to others using lysozyme, glycine or penicillin as cell wall weakening agents are discussed.At the moment we are trying to extend this novel method to other less transformable Lactobacillus strains such as L. plantarum, sakei and curvatus.