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dc.contributor.authorRudge, Timothyen
dc.contributor.authorBrown, James Roberten
dc.contributor.authorFederici, Fernanen
dc.contributor.authorDalchau, Neilen
dc.contributor.authorPhillips, Andrewen
dc.contributor.authorAjioka, Jamesen
dc.contributor.authorHaseloff, Jimen
dc.date.accessioned2015-12-08T16:44:54Z
dc.date.available2015-12-08T16:44:54Z
dc.date.issued2015-10-05en
dc.identifier.citationRudge et al. ACS Synthetic Biology (2015). doi: 10.1021/acssynbio.5b00116en
dc.identifier.issn2161-5063
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/252902
dc.description.abstractAccurate characterization of promoter behavior is essential for the rational design of functional synthetic transcription networks such as logic gates and oscillators. However, transcription rates observed from promoters can vary significantly depending on the growth rate of host cells and the experimental and genetic context of measurement. Further, in vivo measurement methods must accommodate variation in translation, protein folding and maturation rates of reporter proteins, as well as metabolic load. The external factors affecting transcription activity may be considered extrinsic, and the goal of characterization should be to obtain quantitative measures of the intrinsic characteristics of promoters. We have developed a promoter characterization method that is based on a mathematical model for cell growth and reporter gene expression and exploits multiple in vivo measurements to compensate for variation due to extrinsic factors. First, we used optical density and fluorescent reporter gene measurements to account for the effect of differing cell growth rates. Second, we compared the output of reporter genes to that of a control promoter using concurrent dual-channel fluorescence measurements. This allowed us to derive a quantitative promoter characteristic (ρ) that provides a robust measure of the intrinsic properties of a promoter, relative to the control. We imposed different extrinsic factors on growing cells, altering carbon source and adding bacteriostatic agents and demonstrated that the use of ρ values reduced the fraction of variance due to extrinsic factors from 78% to less than 4%. This is a simple and reliable method for quantitative description of promoter properties.
dc.description.sponsorshipTJR was supported by a Microsoft Research studentship and EC FP7 Project No. 612146 (PLASWIRES) awarded to JH, JRB by a Microsoft Research studentship and internship, and FF by CONICYT-PAI/Concurso Nacional de Apoyo al Retorno de Investigadores/as desde el Extranjero Folio 8213002 7, and EPSRC grant EP/H019162/1 awarded to JH. JWA acknowledges the EPSRC and the Wellcome Trust for support.
dc.languageEnglishen
dc.language.isoenen
dc.publisherACS
dc.subjectpromoteren
dc.subjecttranscriptionen
dc.subjectcharacterizationen
dc.subjectratiometricen
dc.subjectfluorescenceen
dc.subjectquantificationen
dc.subjectdesignen
dc.subjectmodelingen
dc.titleCharacterization of intrinsic properties of promotersen
dc.typeArticle
dc.description.versionThis is the author accepted manuscript. The final version is available from ACS via http://dx.doi.org/10.1021/acssynbio.5b00116en
prism.publicationDate2015en
prism.publicationNameACS Synthetic Biologyen
dc.rioxxterms.funderEPSRC
dc.rioxxterms.funderWellcome Trust
dc.rioxxterms.funderEP/H019162/1
rioxxterms.versionofrecord10.1021/acssynbio.5b00116en
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserveden
rioxxterms.licenseref.startdate2015-10-05en
dc.contributor.orcidHaseloff, Jim [0000-0003-4793-8058]
dc.identifier.eissn2161-5063
rioxxterms.typeJournal Article/Reviewen
pubs.funder-project-idBBSRC (BB/L014130/1)
pubs.funder-project-idEPSRC (EP/H019162/1)
pubs.funder-project-idEC FP7 CP (612146)
rioxxterms.freetoread.startdate2016-10-05


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