Abstract
We developed a generic method for high-throughput cloning in bacteria that are less amenable to conventional DNA manipulations. The method involves ligation-independent cloning in an intermediary Escherichia coli vector, which is rapidly converted via vector-backbone exchange (VBEx) into an organism-specific plasmid ready for high-efficiency transformation. We demonstrated VBEx proof of principle for Lactococcus lactis, but the method can be adapted to all organisms for which plasmids are available.
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Acknowledgements
We acknowledge D.J. Slotboom for critical reading of the manuscript, G.K. Schuurman-Wolters, C. Mulligan and D.J.S. for independent testing of the LIC-VBEx method, and the EU-FP6 programme (E-MeP; 504601) for funding.
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E.R.G. designed and performed the experiments and analyzed the data; B.P. supervised the project; E.R.G. and B.P. wrote the manuscript.
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B.P. and E.R.G. have filed an application for a patent for VBEx.
Supplementary information
Supplementary Text and Figures
Supplementary Figure 1, Supplementary Tables 1–2, Supplementary Methods, Supplementary Note. (PDF 112 kb)
Supplementary Data
Frequency and sequence of 3′ extensions of SfiI sites in pro- and eukaryotic genomes. (XLS 470 kb)
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Geertsma, E., Poolman, B. High-throughput cloning and expression in recalcitrant bacteria. Nat Methods 4, 705–707 (2007). https://doi.org/10.1038/nmeth1073
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DOI: https://doi.org/10.1038/nmeth1073
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