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A new logic for DNA engineering using recombination in Escherichia coli

Nature Genetics volume 20, pages 123–128 (1998)Cite this article

Abstract

A straightforward way to engineer DNA in E. coli using homologous recombination is described. The homologous recombination reaction uses RecE and RecT and is transferable between E. coli strains. Several target molecules were manipulated, including high copy plasmids, a large episome and the E. coli chromosome. Sequential steps of homologous or site-specific recombination were used to demonstrate a new logic for engineering DNA, unlimited by the disposition of restriction endonuclease cleavage sites or the size of the target DNA.

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Figure 1: Identification and characterization of a linear plus circular homologous recombination reaction.
Figure 2: Manipulation of a large E. coli episome by multiple recombination steps.
Figure 3: Manipulation of the E. coli chromosome.
Figure 4: Two rounds of ET cloning to introduce a point mutation.
Figure 5: Transferance of ET cloning into a recBC+ host to modify a large episome.

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Acknowledgements

We wish to thank A.J. Clark and M. Berlyn for providing E. coli strains, I. Blomfield for pIB279, the EMBL oligonucleotide and sequencing services for excellent standards and M. Nichols and P.-O. Angrand for discussions. We also thank A.J.H. Smith, M. Meredyth, R. Aasland and the referees for critical readings of the manuscript. Y.Z. is a recipient of an EMBO fellowship. This work was supported in part by a grant from the Volkswagen Foundation, Program on Conditional Mutagenesis.

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  1. Gene Expression Program, European Molecular Biology Laboratory, Meyerhofstr. 1, Heidelberg, 69117, Germany

    Youming Zhang, Frank Buchholz, Joep P.P. Muyrers & A. Francis Stewart

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Correspondence to A. Francis Stewart.

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Zhang, Y., Buchholz, F., Muyrers, J. et al. A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20, 123–128 (1998). https://doi.org/10.1038/2417

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