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Article: Visualizing the proteome of Escherichia coli: An efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genes

TitleVisualizing the proteome of Escherichia coli: An efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genes
Authors
Issue Date2007
PublisherOxford University Press. The Journal's web site is located at http://nar.oxfordjournals.org/
Citation
Nucleic Acids Research, 2007, v. 35 n. 6 How to Cite?
AbstractTo 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 λ-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. © 2007 The Author(s).
Persistent Identifierhttp://hdl.handle.net/10722/68238
ISSN
2015 Impact Factor: 9.202
2015 SCImago Journal Rankings: 7.458
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorWatt, RMen_HK
dc.contributor.authorWang, Jen_HK
dc.contributor.authorLeong, Men_HK
dc.contributor.authorKung, HFen_HK
dc.contributor.authorCheah, KSEen_HK
dc.contributor.authorLiu, Den_HK
dc.contributor.authorDanchin, Aen_HK
dc.contributor.authorHuang, JDen_HK
dc.date.accessioned2010-09-06T06:02:40Z-
dc.date.available2010-09-06T06:02:40Z-
dc.date.issued2007en_HK
dc.identifier.citationNucleic Acids Research, 2007, v. 35 n. 6en_HK
dc.identifier.issn0305-1048en_HK
dc.identifier.urihttp://hdl.handle.net/10722/68238-
dc.description.abstractTo 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 λ-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. © 2007 The Author(s).en_HK
dc.languageengen_HK
dc.publisherOxford University Press. The Journal's web site is located at http://nar.oxfordjournals.org/en_HK
dc.relation.ispartofNucleic Acids Researchen_HK
dc.rightsNucleic Acids Research. Copyright © Oxford University Press.en_HK
dc.subject.meshChromosomes, Bacterial - chemistryen_HK
dc.subject.meshDNA, Bacterial - chemistryen_HK
dc.subject.meshEscherichia coli - chemistry - geneticsen_HK
dc.subject.meshEscherichia coli Proteins - analysis - geneticsen_HK
dc.subject.meshFluorescent Dyes - analysisen_HK
dc.subject.meshGenes, Suppressoren_HK
dc.subject.meshLuminescent Proteins - analysis - geneticsen_HK
dc.subject.meshMethionine Adenosyltransferase - analysis - geneticsen_HK
dc.subject.meshMicroscopy, Fluorescenceen_HK
dc.subject.meshProteome - analysis - geneticsen_HK
dc.subject.meshProteomics - methodsen_HK
dc.subject.meshRecombinant Fusion Proteins - analysisen_HK
dc.subject.meshRecombination, Geneticen_HK
dc.subject.meshTransferases - analysis - geneticsen_HK
dc.titleVisualizing the proteome of Escherichia coli: An efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genesen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0305-1048&volume=35&issue=6&spage=1&epage=11&date=2007&atitle=Visualizing+the+proteome+of+Escherichia+coli:+an+efficient+and+versatile+method+for+labeling+chromosomal+coding+DNA+sequences+(CDSs)+with+fluorescent+protein+genes.en_HK
dc.identifier.emailWatt, RM:rmwatt@hku.hken_HK
dc.identifier.emailCheah, KSE:hrmbdkc@hku.hken_HK
dc.identifier.emailHuang, JD:jdhuang@hkucc.hku.hken_HK
dc.identifier.authorityWatt, RM=rp00043en_HK
dc.identifier.authorityCheah, KSE=rp00342en_HK
dc.identifier.authorityHuang, JD=rp00451en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1093/nar/gkl1158en_HK
dc.identifier.pmid17272300en_HK
dc.identifier.scopuseid_2-s2.0-34247897332en_HK
dc.identifier.hkuros128579en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-34247897332&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume35en_HK
dc.identifier.issue6en_HK
dc.identifier.eissn1362-4962-
dc.identifier.isiWOS:000246123600001-
dc.publisher.placeUnited Kingdomen_HK
dc.identifier.f10001088949-
dc.identifier.scopusauthoridWatt, RM=7102907536en_HK
dc.identifier.scopusauthoridWang, J=8895697000en_HK
dc.identifier.scopusauthoridLeong, M=16301615900en_HK
dc.identifier.scopusauthoridKung, HF=7402514190en_HK
dc.identifier.scopusauthoridCheah, KSE=35387746200en_HK
dc.identifier.scopusauthoridLiu, D=21934191400en_HK
dc.identifier.scopusauthoridDanchin, A=7103235597en_HK
dc.identifier.scopusauthoridHuang, JD=8108660600en_HK
dc.identifier.citeulike1265564-

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