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Plasmid. 2009 Sep;62(2):88-97. doi: 10.1016/j.plasmid.2009.05.002. Epub 2009 May 27.

New yeast recombineering tools for bacteria.

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  • 1Department of Ophthalmology, University of Pittsburgh Eye Center, PA 15213, USA. shanksrm@upmc.edu

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.

PMID:
19477196
[PubMed - indexed for MEDLINE]
PMCID:
PMC2737453
Free PMC Article
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