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MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules

Abstract

We describe a highly engineered in vivo cloning method, mating-assisted genetically integrated cloning (MAGIC), that facilitates the rapid construction of recombinant DNA molecules. MAGIC uses bacterial mating, in vivo site-specific endonuclease cleavage and homologous recombination to catalyze the transfer of a DNA fragment between a donor vector in one bacterial strain and a recipient plasmid in a separate bacterial strain. Recombination events are genetically selected and result in placement of the gene of interest under the control of new regulatory elements with high efficiency. MAGIC eliminates the need for restriction enzymes, DNA ligases, preparation of DNA and all in vitro manipulations required for subcloning and allows the rapid construction of multiple constructs with minimal effort. We show that MAGIC can generate constructs for expression in multiple organisms. As this new method requires only the simple mixing of bacterial strains, it represents a substantial advance in high-throughput recombinant DNA production that will save time, effort and expense in functional genomics studies.

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Figure 1: The MAGIC mating scheme for the production of homologous recombination events through in vivo gene transfer and recombination.
Figure 2: MAGIC cloning efficiencies using counterselective markers.
Figure 3: MAGIC cloning efficiency using the lacO selection.
Figure 4: MAGIC cloning on solid media.
Figure 5: The MAGIC cloning scheme for direct cloning of PCR-generated fragments.
Figure 6: pMAGIC donor vectors.
Figure 7: Expression of ORFs in E. coli, S. cerevisiae and mammals from recombinant constructs made by MAGIC cloning.

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Acknowledgements

We thank B. Wanner for providing the ara and rha promoters, bacterial strains and advice concerning their use; S. Sandler and S. Lovett for discussions; J.W. Harper and M. Schlabach for comments on the manuscript; A. Liang, R. McDonald and T. Westbrook for help with the transfections; and J. Liao for help in recipient vector construction. This work was supported by a grant from the US National Institutes of Health. S.J.E. is an Investigator with the Howard Hughes Medical Institute.

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Correspondence to Stephen J Elledge.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

96-well mating. (PDF 78 kb)

Supplementary Table 1

Bacterial strains. (PDF 150 kb)

Supplementary Table 2

Primer sequences used in this study. (PDF 104 kb)

Supplementary Table 3

Plasmids. (PDF 51 kb)

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Li, M., Elledge, S. MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules. Nat Genet 37, 311–319 (2005). https://doi.org/10.1038/ng1505

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