This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kondo, J K Articles by McKay, L L Search for Related Content PubMed PubMed Citation Articles by Kondo, J K Articles by McKay, L L Agricola Articles by Kondo, J K Articles by McKay, L L

 Previous Article  |  Next Article 

Appl Environ Microbiol. 1984 August; 48(2): 252-259

Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning.

ABSTRACT

The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.

This article has been cited by other articles:

• Luo, H., Wan, K., Wang, H. H. (2005). High-Frequency Conjugation System Facilitates Biofilm Formation and pAM{beta}1 Transmission by Lactococcus lactis. Appl. Environ. Microbiol. 71: 2970-2978 [Abstract] [Full Text]