A suite of Gateway^® cloning vectors for high‐throughput genetic analysis in Saccharomyces cerevisiae

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 galactoseinducible 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).

Bricks and blueprints: methods and standards for DNA assembly

“…Integrases are employed by the popular commercial Gateway cloning method 38 , one of the earliest DNA assembly standard, which uses the lambda integrase in vitro to catalyse directional cloning of DNA parts that are flanked by orthogonal versions of the lambda attB and attP sites (Figure 2a). This method is simple, efficient and reliable, and is widely used for the generation of clone libraries and for expression analysis in eukaryotic systems 39,40 . By synthetically generating four additional orthogonal att recombination sequences, Gateway has also recently been expanded to enable the cloning of multiple parts simultaneously 41 .…”

The protein quality control system manages plant defence compound synthesis

“…For recombinant protein production, the sequences were recombined in the pDEST15 expression vector, and the resulting clones transformed to E. coli BL21 (DE3) cells. For the yeast complementation and localization assays, the pAG426GPD vector 36 was used as the destination vector. To generate bait proteins for immunoprecipitation, the ORFs were fused either N-or C-terminally to the protein G-Streptavidin (GS) tag by Gateway recombination as described 37 .…”

Profiling human protein degradome delineates cellular responses to proteasomal inhibition and reveals a feedback mechanism in regulating proteasome homeostasis

“…The plasmid, pAG426GAL-attR1-ccdB-attR2-EGFP, originally constructed in Dr Lindquist's lab at Massachusetts Institute of Technology [71], was obtained from Addgene, Inc. EGFP was replaced with DNA fragments coding mEGFP, double FLAG epitopes, human ubiquitin with all lysines substituted with arginines, mRFP and double FLAG epitopes to generate mEGFP-(FLAG) 2 -Ub K0 -mRFP-(FLAG) 2 (mEGFP fu -mRFP f ). Then, the fragment containing attR1-ccdB-attR2-mEGFP fu -mRFP f was subcloned into the lentivirus vector, pCDH-CMV-MCS-EF1a-Puro (System Biosciences), using XbaI and NheI restriction sites, to obtain the parental vector, pCDH-ccdB-mEGFP fu -mRFP f , for mammalian expression.…”