Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1993 Jun;74(6):629-36.
doi: 10.1111/j.1365-2672.1993.tb05195.x.

Improved cloning vectors and transformation procedure for Lactococcus lactis

J M Wells  1 , P W Wilson, R W Le Page
Affiliations

Improved cloning vectors and transformation procedure for Lactococcus lactis

J M Wells et al. J Appl Bacteriol. 1993 Jun.

Abstract

Four shuttle vectors (pMIG 1, 2, 2H and 3) have been constructed based on the broad host-range plasmid pCK1. All the pMIG vectors possess a multiple cloning site containing 12 or more unique restriction enzyme sites, and are stably maintained at either high or low copy number in Lactococcus lactis and in Escherichia coli. By cloning the E. coli pUC replicon into one of these vectors a plasmid was constructed which can replicate to high copy number in recA strains of E. coli. The broad host-range of the pCK1 replicon may enable these cloning vectors to be used in a number of Gram-positive bacteria. One of these vectors was used to optimize an electroporation procedure for transformation of a commonly used plasmid-cured strain MG1363 of L. lactis which routinely yielded 1 x 10(7) to 5 x 10(7) transformants micrograms-1 supercoiled DNA using stored, snap-frozen cells. This transformation efficiency was obtained by growing the cells in medium containing the cell wall weakening agent glycine, to an upper limit of 2.5% w/v. Although growth of L. lactis strain MG1363 was inhibited by the use of 0.5 mol l-1 sucrose as an osmotic stabilizer, the presence of sucrose in the electroporation buffer was critical for high transformation efficiency. Other variables which were tested for their effect on the efficiency of transformation were cell concentration, DNA concentration, pulse time and field strength. These results provide a model procedure which can be followed to optimize conditions for the genetic transformation of various strains of L. lactis.

PubMed Disclaimer

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

Publication types

LinkOut - more resources

-