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Nucleic Acids Research Advance Access originally published online on June 21, 2008
Nucleic Acids Research 2008 36(14):e84; doi:10.1093/nar/gkn359
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Nucleic Acids Research, 2008, Vol. 36, No. 14 e84
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Rapid and efficient construction of markerless deletions in the Escherichia coli genome

Byung Jo Yu1, Kui Hyeon Kang1, Jun Hyoung Lee1, Bong Hyun Sung1, Mi Sun Kim2 and Sun Chang Kim1,*

1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701 and 2Biomass Team, Korea Institute of Energy Research, Daejeon, 305-343, Korea

*To whom correspondence should be addressed. Tel: +82 42 869 2619; Fax: +82 42 869 2610; Email: sunkim{at}kaist.ac.kr

Received March 31, 2008. Revised May 7, 2008. Accepted May 20, 2008.

We have developed an improved and rapid genomic engineering procedure for the construction of custom-designed microorganisms. This method, which can be performed in 2 days, permits restructuring of the Escherichia coli genome via markerless deletion of selected genomic regions. The deletion process was mediated by a special plasmid, pREDI, which carries two independent inducible promoters: (i) an arabinose-inducible promoter that drives expression of {lambda}-Red recombination proteins, which carry out the replacement of a target genomic region with a marker-containing linear DNA cassette, and (ii) a rhamnose-inducible promoter that drives expression of I-SceI endonuclease, which stimulates deletion of the introduced marker by double-strand breakage-mediated intramolecular recombination. This genomic deletion was performed successively with only one plasmid, pREDI, simply by changing the carbon source in the bacterial growth medium from arabinose to rhamnose. The efficiencies of targeted region replacement and deletion of the inserted linear DNA cassette were nearly 70 and 100%, respectively. This rapid and efficient procedure can be adapted for use in generating a variety of genome modifications.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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