Nucleic Acids Research Advance Access originally published online on January 31, 2007
Nucleic Acids Research 2007 35(6):e37; doi:10.1093/nar/gkl1158
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Nucleic Acids Research, 2007, Vol. 35, No. 6 e37
© 2007 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 |
Visualizing the proteome of Escherichia coli: an efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genes
1Open Laboratory of Chemical Biology, The Institute of Molecular Technology for Drug Discovery and Synthesis, Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China, 2Department of Biochemistry, The University of Hong Kong, 3/F Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong SAR, China, 3The Center for Emerging Infectious Diseases, Faculty of Medicine, Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, China, 4National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College (PUMC), Beijing 100005, P.R. China, 5Unité GGB, CNRS URA 2171, Institut Pasteur, 28 rue Dr. Roux, 75015 Paris, France and 6HKU-Pasteur Research Centre, Dexter HC Man Building, 8, Sassoon Road, Pokfulam, Hong Kong SAR, China
*To whom correspondence should be addressed. Tel: (+852) 2819 2810; Fax: (+852) 2855 1254; E-mail: jdhuang{at}hkucc.hku.hk
Correspondence may also be addressed to Antoine Danchin. Tel: (+331) 45 68 84 42; Fax: (+331) 45 68 89 48; E-mail: adanchin{at}pasteur.fr
Received June 27, 2006. Revised December 15, 2006. Accepted December 19, 2006.
To investigate the feasibility of conducting a genomic-scale protein labeling and localization study in Escherichia coli, a representative subset of 23 coding DNA sequences (CDSs) was selected for chromosomal tagging with one or more fluorescent protein genes (EGFP, EYFP, mRFP1, DsRed2). We used 
-Red recombination to precisely and efficiently position PCR-generated DNA targeting cassettes containing a fluorescent protein gene and an antibiotic resistance marker, at the C-termini of the CDSs of interest, creating in-frame fusions under the control of their native promoters. We incorporated cre/loxP and flpe/frt technology to enable multiple rounds of chromosomal tagging events to be performed sequentially with minimal disruption to the target locus, thus allowing sets of proteins to be co-localized within the cell. The visualization of labeled proteins in live E. coli cells using fluorescence microscopy revealed a striking variety of distributions including: membrane and nucleoid association, polar foci and diffuse cytoplasmic localization. Fifty of the fifty-two independent targeting experiments performed were successful, and 21 of the 23 selected CDSs could be fluorescently visualized. Our results show that E. coli has an organized and dynamic proteome, and demonstrate that this approach is applicable for tagging and (co-) localizing CDSs on a genome-wide scale.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.