))sors - ghent university academic bibliography · introduction "if you cannot measure it, you...
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Introduction
"If you cannot measure it, you cannot improve it."g SLordgKelvinIgB@thgcenturyAOgMeasurementgofgkeygpathwaygmetabolitesg isgessentialg ingmetabolicgengineeringg togensuregthegdevelopmentgofgefficientgandgoptimalgmicrobialgproductiongstrainsOg/urrentgmethodsgforganalysisgofgintracellulargmetabolitesgaregcumbersomegandgdemandgexgvivoIgdestructivegtechniquesgsuchgasgHPL/IgG/gandgL/&MSOg:sgangelegantgsolutionIg transcriptionalgbiosensorsIgconsistinggofg transcriptionalgregulatorygcircuitsgfoundg ingnatureIg cangbegusedOgThesegbiosensorsgenableg ing vivoIg non&destructiveIg proportionalIg high&throughputgandgevengsimultaneousgmeasurementgofg specificgmetabolitesgofginterestO
Thegauthorsgwishgtogthankgthegx:gentschapgInnoverengGgOndernemenxgingFlandersgS:IOAgforgfinancialgsupportgingthegframeworkgofgthegPhODOggrantgofgWrechtgDegPaepeOgThisgresearchgisgalsogsupportedgbygthegMultidisciplinarygResearchgPartnershipgGhentgWio&Economygandgtheg/ondexgprojectgSGODERBOBENAOg
WrechtODePaepe’UGentObeLaboratorygofgIndustrialgWiotechnologygandgWiocatalysis
DepartmentgofgWiochemicalgandgMicrobialgTechnologyGhentgUniversityIg/oupuregLinksg30EIg@DDDgGhentg&gWelgiumg
Acknowledgments Contact
Tailor-made)Transcriptional)BiosensorsWrechtgDegPaepeBIgJogMaertensBIgWartelgVanholmeRgandgMarjangDegMeyB
BMEMOg&gMetabolicgEngineeringgGroupg&gLaboratorygofgIndustrialgWiotechnologygandgWiocatalysisg&gGhentgUniversityg&gWelgiumRVIWg&gVlaamsgInstituutgvoorgWiotechnologieg&gDepartmentgofgPlantgSystemsgWiologyg&gWegliumg
Tailor-made)characteristic)response)curve
Transcription)factor Transcription)factor)binding)sites
Tailor-made)substrate)specificity
Biosensor)copy)number
Chimeric)transcriptional)regulation)pairs Chimeric)transcription)factors
Current)results
Theg ratiog ofg transcriptiong factorg expressiong versusg theg copyg numberg ofg theg bindingg sitesg andg thegintracellulargconcentrationgofgthegspecificgligandgplaysgagkeygrolegingdefininggthegbiosensorgresponsegcurveOgWygalteringgthegpromotergandgRWSgstrengthgofgthegtranscriptiongfactorgoperonIgthegresponsegcurvegcangbegalteredO
Engineeringgthegnativegbiosensorgpartsgenablesgthegdevelopmentgofguniquegbiosensorsgwithgcharacteristicgresponsegcurvesgcustomizedgtogthegresearcher’sgneedsOgTranscriptionalg biosensorg architectureg cang beg dividedg intog threeg basicg levelsg ofg controlUg (1)g theg transcriptiong factorg operonIg (2)g theg reporterg operong withg thegtranscriptiongfactorgbindinggsitesgandg(3)gthegSplasmidAgcopygnumbergofgthegcompletegbiosensorOgggg
Engineeringgtranscriptiongfactorsgenablesgthegdevelopmentgofgbiosensorsgwithguniquegmolecule&specificitiesgcustomizedgtogthegresearcher’sgneedsOgWithingthegsamegtranscriptiongfactorgfamilyIghighlygconservedgDN:&gandgligand&bindinggdomainsgaregdistinguishableOg:dditionallyIgthegcorrespondinggtranscriptiongfactorgbindinggsitesgalsog showg highg conservationOg ThereforeIg completeg transcriptiong factorsg org eveng specificg ligand&bindingg proteing domainsg areg interchangedg withg ag Spartg ofAg thegtranscriptiongfactorgofgagcharaterizedgbiosensorgtogcreategchimericgbiosensorsgwithgothergligandgspecificitiesgbutgpredictablegresponsegcurvesO
FluorescentproteinPromoterTranscriptionfactorPromoter
ThegSplasmidAgcopygnumbergofgthegbiosensorgdirectlygeffectsgthegtotalgexpressiongofgbothgtranscriptiongfactorgandgfluorescentgreportergproteingandgthegcopygnumbergofgthegtranscriptiongfactorgbindinggsitesOg/onsequentlyIgthegbiosensorgcopygnumbergco&definesgthegbiosensorgresponsegcurveOgggg
Dueg tog theghighgconservationgofgDN:&bindinggsitesgacrossgdifferentgspeciesIgcombininggag ligandgspecificgtranscriptiongfactorgfromgonegspeciesgwithgDN:&bindinggsitesgfromgagcharacterizedgbiosensorIgcangcreategagfunctionalg chimericg bioesensorg withg differentg ligandg specificityOg Theseg chimericg biosensorsg onlyg differg ingtheirgtranscriptiongfactorIgthusgenhancinggconstructiongefficiencygandgresponsegcurvegpredictabilityO
Transcriptiong factorsg withg conservedg andg distinctg DN:&g andg ligand&bindingg proteing domainsg andg withgdifferentgligandgspecificitiesgaregusedgtogconstructgchimericgtranscriptiongfactorsOgThereforeIgagcharacterizedgbiosensorg withg ag definedg responseg curveg isg usedg asg ag chassisg forg constructingg ag predictableg chimericgbiosensorgwithgagchimericgtranscriptiongfactorgthatgcontainsgthegsubstrategbindinggdomaingwithgthegdesiredgspecificityOg
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LigandgconcentrationgSmgFLA
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Step)1:)MimickinggnativegpromotergandgRWSgstrengthgwithgsyntheticgpromotergandgRWSggg
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