A suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae - PubMed (original) (raw)

A suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae

Simon Alberti et al. Yeast. 2007 Oct.

Abstract

In the post-genomic era, academic and biotechnological research is increasingly shifting its attention from single proteins to the analysis of complex protein networks. This change in experimental design requires the use of simple and experimentally tractable organisms, such as the unicellular eukaryote Saccharomyces cerevisiae, and a range of new high-throughput techniques. The Gateway system has emerged as a powerful high-throughput cloning method that allows for the in vitro recombination of DNA with high speed, accuracy and reliability. Two Gateway-based libraries of overexpression plasmids containing the entire complement of yeast open reading frames (ORFs) have recently been completed. In order to make use of these powerful resources, we adapted the widely used pRS series of yeast shuttle vectors for use in Gateway-based cloning. The resulting suite of 288 yeast Gateway vectors is based upon the two commonly used GPD and GAL1 promoter expression systems that enable expression of ORFs, either constitutively or under galactose-inducible conditions. In addition, proteins of interest can be fused to a choice of frequently used N- or C-terminal tags, such as EGFP, ECFP, EYFP, Cerulean, monomeric DsRed, HA or TAP. We have made this yeast Gateway vector kit available to the research community via the non-profit Addgene Plasmid Repository (http://www.addgene.org/yeast\_gateway).

Copyright 2007 John Wiley & Sons, Ltd.

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Figures

Figure 1

Figure 1. Overview of Gateway system and Generation of pAG yeast destination vectors

A) The Gateway system of recombination-based cloning involves an LR reaction, in which an entry clone, containing a gene of interest is mixed with a destination vector, containing features of interest (e.g. promoter, protein tags, etc.). The destination vector harbors a recombination site-flanked bacterial “death” gene (ccdB) that is exchanged for the gene of interest contained in the entry clone. Transformation of E. coli that are sensitive to the ccdB effects allows for selection of expression clones. This reaction is reversible (BP reaction); expression clones can be used to re-generate entry clones. B) The set of pAdvancedGateway destination vectors allows for a choice of constitutive (GPD) or inducible (GAL1) promoters, N- or C-terminal protein tags, integrating or extra-chromosomal origins of replication, high- (2μ) or low-copy number (CEN), as well as a choice of auxotrophic markers. Caution: Since these destination vectors contain the ccdB cassette, they must be propagated in ccdB-resistant E. coli (e.g. DB3.1).

Figure 2

Figure 2. Practical application of Advanced Gateway vectors to experiments with α–synuclein in yeast

A) An entry clone containing human α–synuclein (with or without stop codon) was used in 1 hour in vitro LR reactions to generate untagged, EGFP-, DsRed-, HA-, or TAP-tagged expression constructs. The Gateway-generated fluorescently tagged proteins localized properly to the plasma membrane (B), whereas EGFP alone was distributed throughout the cytoplasm; the untagged protein induced cellular toxicity (C) and immunoblot analysis detected the appropriate size tagged proteins (D). The asterisk (*) in D indicates a non-specific immunoreactive band resulting from the Protein A portion of the TAP tag.

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