))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." SLord KelvinI B@th centuryAO Measurement of key pathway metabolites is essential in metabolic engineering to ensure the development of efficient and optimal microbial production strainsO /urrent methods for analysis of intracellular metabolites are cumbersome and demand ex vivoI destructive techniques such as HPL/I G/ and L/&MSO :s an elegant solutionI transcriptional biosensorsI consisting of transcriptional regulatory circuits found in natureI can be usedO These biosensors enable in vivoI non&destructiveI proportionalI high&throughput and even simultaneous measurement of specific metabolites of interestO The authors wish to thank the x:gentschap Innoveren G Ondernemenx in Flanders S:IOA for financial support in the framework of the PhODO grant of Wrecht De PaepeO This research is also supported by the Multidisciplinary Research Partnership Ghent Wio&Economy and the /ondex project SGODE2BOBENAO WrechtODePaepe’UGentObe Laboratory of Industrial Wiotechnology and Wiocatalysis Department of Wiochemical and Microbial Technology Ghent UniversityI /oupure Links 30EI @DDD Ghent & Welgium Acknowledgments Contact Tailor-made Transcriptional Biosensors Wrecht De Paepe B I Jo Maertens B I Wartel Vanholme 2 and Marjan De Mey B B MEMO & Metabolic Engineering Group & Laboratory of Industrial Wiotechnology and Wiocatalysis & Ghent University & Welgium 2 VIW & Vlaams Instituut voor Wiotechnologie & Department of Plant Systems Wiology & Weglium 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 The ratio of transcription factor expression versus the copy number of the binding sites and the intracellular concentration of the specific ligand plays a key role in defining the biosensor response curveO Wy altering the promoter and RWS strength of the transcription factor operonI the response curve can be alteredO Engineering the native biosensor parts enables the development of unique biosensors with characteristic response curves customized to the researcher’s needsO Transcriptional biosensor architecture can be divided into three basic levels of controlU (1) the transcription factor operonI (2) the reporter operon with the transcription factor binding sites and (3) the SplasmidA copy number of the complete biosensorO Engineering transcription factors enables the development of biosensors with unique molecule&specificities customized to the researcher’s needsO Within the same transcription factor familyI highly conserved DN:& and ligand&binding domains are distinguishableO :dditionallyI the corresponding transcription factor binding sites also show high conservationO ThereforeI complete transcription factors or even specific ligand&binding protein domains are interchanged with a Spart ofA the transcription factor of a charaterized biosensor to create chimeric biosensors with other ligand specificities but predictable response curvesO Fluorescent protein Promoter Transcription factor Promoter The SplasmidA copy number of the biosensor directly effects the total expression of both transcription factor and fluorescent reporter protein and the copy number of the transcription factor binding sitesO /onsequentlyI the biosensor copy number co&defines the biosensor response curveO Due to the high conservation of DN:&binding sites across different speciesI combining a ligand specific transcription factor from one species with DN:&binding sites from a characterized biosensorI can create a functional chimeric bioesensor with different ligand specificityO These chimeric biosensors only differ in their transcription factorI thus enhancing construction efficiency and response curve predictabilityO Transcription factors with conserved and distinct DN:& and ligand&binding protein domains and with different ligand specificities are used to construct chimeric transcription factorsO ThereforeI a characterized biosensor with a defined response curve is used as a chassis for constructing a predictable chimeric biosensor with a chimeric transcription factor that contains the substrate binding domain with the desired specificityO 0 10 20 30 0 50 100 150 0 10 20 30 0 50 100 150 0 10 20 30 0 50 100 150 Step 1: Mimicking native promoter and RWS strength with synthetic promoter and RWS Step 2: :ltered promoter and RWS strength for altered response curves The ratio of fluorescent protein expression versus the copy number of the binding sites and the expression of transcription factor plays a key role in defining the biosensor response curveO Wy altering the promoter and RWS strength of the reporter operon and the position and copy number of the transcription factor binding sitesI the response curve can be alteredO 0 10 20 30 0 50 100 150 Ligand concentration SmgFLA Ligand concentration SmgFLA 0 5 10 15 20 25 0 25 Ligand concentration Omg/LD IPTG OμMD 0 2 4 8 Copy number Relative fluorescence/OD600 O-D Relative fluorescence/OD600 O-D 0 10 20 30 0 50 100 150 0 10 20 30 0 50 100 150 0 10 20 30 0 50 100 150 Step 1: Mimicking native promoter and RWS strength with synthetic promoter and RWS Step 2: :ltered promoter and RWS strength and altered transcription factor binding sites for altered response curves Ligand concentration SmgFLA Current results Perspectives Relative fluorescence/OD600 O-D Perspectives 0 10 20 30 0 50 100 150 Perspectives Relative fluorescence/OD600 O-D The use of a plasmid with an inducible ori enables varying the plasmid copy number proportional to the IPTG concentrationO /onsequentlyI the effect of copy number on the characteristic response curve can be investigatedO Current results Ligand concentration Omg/LD Relative fluorescence/OD600 O-D Fluorescent protein Transcription factor Transcription factor Fluorescent protein Binding sites Binding sites RBS RBS Binding sites Promoter RBS Promoter RBS Promoter RBS Promoter RBS Transcription factor 1 DNA-binding domain Substrate-binding domain Chimeric transcription factor 1-2 Transcription factor 2 DNA-binding domain Substrate-binding domain DNA-binding domain Substrate-binding domain Fluorescent protein Binding sites Promoter RBS Ligand concentration SmgFLA Ligand concentration SmgFLA
Transcript of ))sors - Ghent University Academic Bibliography · Introduction "If you cannot measure it, you...
Introduction
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