New yeast recombineering tools for bacteria

doi:10.1016/j.plasmid.2009.05.002

Plasmid

Volume 62, Issue 2, September 2009, Pages 88-97

https://doi.org/10.1016/j.plasmid.2009.05.002 Get rights and content

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.

Introduction

Cloning by Saccharomyces cerevisiae homologous recombination is a powerful technique that has been harnessed for bacterial applications (Raymond et al., 2002a, Shanks et al., 2006). A “gap” or double stranded break (DSB) in a vector capable of replication in yeast can be repaired through homologous recombination with PCR-generated amplicons, because this repair event can be selected for with markers on the vector backbone the amplicon can be unmarked, i.e. have no selectable marker (DeMarini et al., 2001, Jansen et al., 2005, Mallet and Jacquet, 1996, Oldenburg et al., 1997, Orr-Weaver and Szostak, 1983). The advantages cloning with of yeast recombination are (A) that multiple unmarked pieces of DNA can be sewn together seamlessly without multiple rounds of amplification and without restriction sites on the ends, (B) the site of recombination in a vector is not limited to a cut junction and can be 100s of base pairs away, and (C) the method is efficient, robust and simple to perform. Yeast recombination can require longer overall time than traditional in vitro due to the growth rate of S. cerevisiae; however, the hands-on experimental time requirements are minimal.

Here, we report two new tools that take advantage of yeast recombineering. The first is a method to make targeted deletions and short insertions in cloned genes using single stranded DNA oligomers. The second is a new set of bacteria–yeast shuttle vectors incorporating a number of replicons, promoters and selectable markers for a variety of uses in Gram-positive and Gram-negative organisms, which can be utilized for recombineering with budding yeast.

Section snippets

Microbes and growth conditions

Bacterial and yeast strains used in this study are listed in Table 1. All bacteria were cultured using LB medium (Bertani, 1951). S. cerevisiae was cultured with YPD broth (1% Bacto-yeast extract, 2% Bacto-peptone and 2% dextrose), and selections were done using uracil drop-out medium (SC-ura) (Burke and Stearns, 2000). For β-galactosidase (β-gal) activity determination, SC-ura medium was modified by the addition of 80 μg/ml X-gal, was phosphate buffered (0.7% dibasic sodium phosphate and 0.3%

Directed deletions and insertions without PCR

A previous report elegantly showed that single stranded DNA (ssDNA) oligomers could be used to target recombination between a double stranded DNA (dsDNA) amplicon and a dsDNA episome using S. cerevisiae recombination (Raymond et al., 2002b). We hypothesized that ssDNA oligomers could be used to make targeted deletions in cloned genes using S. cerevisiae recombination. This would be very convenient as purchased oligomers could be used immediately, with no PCR steps, to target specific deletions

Discussion

This report describes both the use of S. cerevisiae-based recombineering to generate oligonucleotide-mediated deletions and insertions and an expanded vector tool-box. A previous group of yeast-recombineering vectors published by our group were useful for gene expression in a wide array of Gram-negative bacteria, but the allelic replacement vectors were focused for Pseudomonas species (Shanks et al., 2006). This current vector set expands on the previous set, and includes shuttle vectors that

Acknowledgment

We are grateful to Nicholas Stella and Christina Medaglia for technical assistance, and Eric Rubin, Frederic Boccard, Patrick Stragier, Dennis Cvitkovitch, Paul Kinchington, Eric Lambie, Karen Skorulpski, Victor Ambrose, Joseph Horzempa, the S.U.B., and Ambrose Cheung labs for providing plasmids, strains, ideas and restriction enzymes. This work was supported by NIH R21-AI055774 Grant to G.A.O., NIH fellowship F32 GM66658-01A1 and Research to Prevent Blindness Career Development Award to

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View full text

New yeast recombineering tools for bacteria

Abstract

Introduction

Section snippets

Microbes and growth conditions

Directed deletions and insertions without PCR

Discussion

Acknowledgment

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Methods Enzymol.

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Curr. Opin. Biotechnol.

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Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from Gram-positive organisms

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DNA and protein interactions in the regulation of plasmid replication

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