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Nucleic Acids Research Advance Access published online on July 18, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn449
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© 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

Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli

Ju Young Lee1, Bong Hyun Sung1, Byung Jo Yu1, Jun Hyoung Lee1, Sang Hee Lee1, Mi Sun Kim2, Michael D. Koob3 and Sun Chang Kim1,*

1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 2Biomass Team, Korea Institute of Energy Research, Daejeon, Korea and 3Department of Laboratory Medicine and Pathology, Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USA

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

Received November 12, 2007. Revised June 19, 2008. Accepted June 27, 2008.

Now that many genomes have been sequenced and the products of newly identified genes have been annotated, the next goal is to engineer the desired phenotypes in organisms of interest. For the phenotypic engineering of microorganisms, we have developed novel artificial transcription factors (ATFs) capable of reprogramming innate gene expression circuits in Escherichia coli. These ATFs are composed of zinc finger (ZF) DNA-binding proteins, with distinct specificities, fused to an E. coli cyclic AMP receptor protein (CRP). By randomly assembling 40 different types of ZFs, we have constructed more than 6.4 x 104 ATFs that consist of 3 ZF DNA-binding domains and a CRP effector domain. Using these ATFs, we induced various phenotypic changes in E. coli and selected for industrially important traits, such as resistance to heat shock, osmotic pressure and cold shock. Genes associated with the heat-shock resistance phenotype were then characterized. These results and the general applicability of this platform clearly indicate that novel ATFs are powerful tools for the phenotypic engineering of microorganisms and can facilitate microbial functional genomic studies.


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