Open Access Highly Accessed Methodology article

A MultiSite GatewayTM vector set for the functional analysis of genes in the model Saccharomyces cerevisiae

Astrid Nagels Durand1,2, Tessa Moses1,2,3,4, Rebecca De Clercq1,2, Alain Goossens1,2*† and Laurens Pauwels1,2†

Author Affiliations

1 Department of Plant Systems Biology, VIB, Technologiepark 927, B-9052, Gent, Belgium

2 Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, B-9052, Ghent, Belgium

3 Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 31, B-3001, Leuven-Heverlee, Belgium

4 Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001, Leuven-Heverlee, Belgium

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BMC Molecular Biology 2012, 13:30 doi:10.1186/1471-2199-13-30

Published: 20 September 2012

Abstract

Background

Recombinatorial cloning using the GatewayTM technology has been the method of choice for high-throughput omics projects, resulting in the availability of entire ORFeomes in GatewayTM compatible vectors. The MultiSite GatewayTM system allows combining multiple genetic fragments such as promoter, ORF and epitope tag in one single reaction. To date, this technology has not been accessible in the yeast Saccharomyces cerevisiae, one of the most widely used experimental systems in molecular biology, due to the lack of appropriate destination vectors.

Results

Here, we present a set of three-fragment MultiSite GatewayTM destination vectors that have been developed for gene expression in S. cerevisiae and that allow the assembly of any promoter, open reading frame, epitope tag arrangement in combination with any of four auxotrophic markers and three distinct replication mechanisms. As an example of its applicability, we used yeast three-hybrid to provide evidence for the assembly of a ternary complex of plant proteins involved in jasmonate signalling and consisting of the JAZ, NINJA and TOPLESS proteins.

Conclusion

Our vectors make MultiSite GatewayTM cloning accessible in S. cerevisiae and implement a fast and versatile cloning method for the high-throughput functional analysis of (heterologous) proteins in one of the most widely used model organisms for molecular biology research.

Keywords:
Gateway cloning; MultiSite; Saccharomyces cerevisiae; Yeast; Vector; Fusion protein; Epitope tag; Jasmonate; Arabidopsis thaliana