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New yeast recombineering tools for bacteria.

ABSTRACT: Recombineering with Saccharomyces cerevisiae is a powerful methodology that can be used to clone multiple unmarked pieces of DNA to generate complex constructs with high efficiency. Here, we introduce two new tools that utilize the native recombination enzymes of S. cerevisiae to facilitate the manipulation of DNA. First, yeast recombineering was used to make directed nested deletions in a bacteria-yeast shuttle plasmid using only one or two single stranded oligomers, thus obviating the need for a PCR step. Second, we have generated several new shuttle vectors for yeast recombineering capable of replication in a wide variety of bacterial genera. As a demonstration of utility, some of the approaches and vectors generated in this study were used to make a pigP deletion mutation in the opportunistic pathogen Serratia marcescens.

SUBMITTER: Shanks RM

PROVIDER: S-EPMC2737453 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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New yeast recombineering tools for bacteria.

Shanks Robert M Q RM Kadouri Daniel E DE MacEachran Daniel P DP O'Toole George A GA

Plasmid 20090527 2

Recombineering with Saccharomyces cerevisiae is a powerful methodology that can be used to clone multiple unmarked pieces of DNA to generate complex constructs with high efficiency. Here, we introduce two new tools that utilize the native recombination enzymes of S. cerevisiae to facilitate the manipulation of DNA. First, yeast recombineering was used to make directed nested deletions in a bacteria-yeast shuttle plasmid using only one or two single stranded oligomers, thus obviating the need for ...[more]

PMID: 19477196

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