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The Selection of Recombinant Binary Plasmids Generated by Gateway® LR Cloning in the Escherichia coli Strain C2110

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Abstract

Gateway® cloning is widely used in molecular biology laboratories. Various binary vectors used for Agrobacterium-mediated plant transformation have been modified as destination vectors that are convenient for the sub-cloning of targeted genes from Entry plasmids. However, when the destination and Entry plasmids have the same antibiotic resistance genes for bacterial selection, the non-recombinant Entry plasmid in the LR reaction mixture can compete with the recombinant destination plasmid during bacterial transformation and selection. Methods for the effective selection of recombinant destination plasmids are highly desirable. In this study, we demonstrated that Escherichia coli strain C2110, which is defective in DNA polymerase I (pAL1), could be used to select a recombinant binary destination plasmid with a RK2 replication origin, while the replication of the Entry plasmid with a ColE1 replication origin was inhibited. Plasmid DNA isolated from C2110 by a traditional mini-prep kit was used for restriction enzyme digestion, DNA sequencing, and Arabidopsis protoplast transfection. The binary plasmid in C2110 was also efficiently mobilized into Agrobacterium tumefaciens via the tri-parental conjugation method.

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Acknowledgments

Financial support was provided by grants from the Binational Agricultural Research and Development Fund (BARD; US-4216-09 to BZ), US NSF (IOS-0845283 to BZ), and the Virginia Agricultural Experiment Station (VA135872).

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

    Shuchi Wu & Bingyu Zhao

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  1. Shuchi Wu
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Correspondence to Bingyu Zhao.

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Wu, S., Zhao, B. The Selection of Recombinant Binary Plasmids Generated by Gateway® LR Cloning in the Escherichia coli Strain C2110. Mol Biotechnol 54, 125–132 (2013). https://doi.org/10.1007/s12033-012-9548-1

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