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Open Access Methodology

A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens

Sy Mamadou Traore and Bingyu Zhao*

Author Affiliations

Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061, USA

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Plant Methods 2011, 7:42  doi:10.1186/1746-4811-7-42

Published: 7 December 2011

Abstract

Background

Cloning genes into plasmid vectors is one of the key steps for studying gene function. Recently, Invitrogen™ developed a convenient Gateway® cloning system based on the site-specific DNA recombination properties of bacteriophage lambda and the cytotoxic protein ccdB, which is lethal to most E. coli strains. The ccdB protein, however, is not toxic to Agrobacterium tumefaciens, an important player often used for studying gene function in planta. This limits the direct application of the Gateway® cloning system in plant transformation-mediated research.

Results

In this study, we constructed a novel Gateway®-compatible destination vector, pEG101-SacB/R, by replacing the ccdB gene with a SacB-SacR gene cassette as the negative selectable marker.

Conclusion

Our results demonstrate that the new pEG101-SacB/R destination vector can be used for Gateway® cloning in Agrobacterium tumefaciens. pEG101-SacB/R will be a valuable tool for high-throughput functional analysis of genes in planta.