Abstract
BackgroundRecombinatorial 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.ResultsHere, 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.ConclusionOur 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.
Highlights
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
Value of the vector set The value of this vector set was exemplified by a yeast three-hybrid (Y3H) experiment, in which we investigated the formation of the ternary protein complex by the Arabidopsis JAZ3, Novel Interactor of JAZ (NINJA), and TPL proteins
We have proposed recently that the adaptor protein NINJA bridges the jasmonate ZIM-domain (JAZ) proteins to TPL proteins and thereby forms a repressor complex that blocks the cellular programs regulated by the jasmonates, ubiquitous plant hormones that regulate various aspects of plant growth, development, and survival [33]
Summary
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. 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. To cope with the large amounts of constructs needed, scientists make use of high-throughput cloning technologies, such as the GatewayTM technology (Invitrogen; http://www.invitrogen. com/), which is based on the site-specific recombination system from the bacteriophage lambda that facilitates A PCR product or B1
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