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. 1986 Mar;165(3):831–836. doi: 10.1128/jb.165.3.831-836.1986

Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector.

R Wirth, F Y An, D B Clewell
PMCID: PMC214503  PMID: 3005240

Abstract

A highly efficient protoplast transformation system for Streptococcus faecalis has been developed by systematically optimizing different parameters. Up to 10(6) transformants per micrograms of DNA were consistently obtained within 3 days, and cell wall regeneration of protoplasts was virtually 100%. A systematic search for useful vectors showed that the broad-host-range plasmid pIP501 could transform S. faecalis at a high frequency (6.3 X 10(4) transformants per microgram). By combining a high-copy-number derivative of pIP501, designated pGB354, with the Escherichia coli vector pACYC184, we constructed a new E. coli-S. faecalis shuttle vector (pAM401) having nine unique restriction sites. In a shotgun cloning experiment, we ligated a tetracycline resistance determinant from Streptococcus sanguis chromosomal DNA into pAM401 by direct transformation of S. faecalis, establishing the utility of the protoplast transformation system and of the new shuttle vector.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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