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ObtainingbindingaffinitiesofCsrAtodifferentuntranslatedregions(UTRs)toimprovemodelforpredictionofCsrA interactions
AlexMoon-WalkerGrantGelderman andAbigailLeistra
Dr.LydiaM.ContrerasDepartmentofChemicalEngineering,TheUniversityofTexasatAustin
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E.coli
GeneralFacts• Gramnegativerod• 4.6millionbasepairgenome• Partofgutflora
E.ColiintheWorldofMetabolicEngineering• Fastgrowth• Relativelysimplebiologicalsystem• AbilitytotakeupplasmidswithforeignDNA
https://c1.staticflickr.com/9/8723/16578744517_ed4293d3e7_b.jpg
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UsingBiologicalSystemsforLargeScalePharmaceuticalProduction
Insulin TherapeuticMonoclonalAntibodies
Erythromycin
https://upload.wikimedia.org/wikipedia/commons/0/0d/InsulinHexamer.jpg http://www.novimmune.com/img/mab-480.jpg https://upload.wikimedia.org/wikipedia/commons/thumb/6/65/Erythromycin_A.svg/2000px-Erythromycin_A.svg.png
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CentralDogmaofBiology
DNARNA Protein
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Ref1
PPP
Foldoverexpression
UseofMetabolicPathwaysforIndustrialLevelProductionofPharmaceuticals
5McKee, Adrienne E et al. "Manipulation Of The Carbon Storage Regulator System For Metabolite Remodeling And Biofuel Production In Escherichia Coli". Microb Cell Fact 11.1 (2012): 79.
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TheCarbonStorageRegulatory(Csr)System
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CsrA bindstocanonicalANGGAsequencesin5’UTRs
A U G C N
ANGGA
CsrA CsrA
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CsrA bindstocanonicalANGGAsequencesin5’UTRs
A U G C N
ANGGA
CsrA CsrA
Controloftranslationalregulationsystemspresentsgreatopportunitiesformetabolicengineering
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CsrA BindingPredictiveModel
UTRStructure
RNASequence
BindingAffinityCalculator
EstimatedFreeEnergy
CurrentProblem:Howaccurateisthemodel’sevaluationandpredictionofCsrAtargets?
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Hypothesis
Estimatedbindingaffinityconstantsfrommodelcapturephysiologically-relevantCsrA-mRNAaffinities.
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UsingGFPasreportertoanalyzechangesingeneexpression
InducedCsrA
Uninduced CsrA
DeoD genetarget
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PlasmidConstructs
9000x5000x2000x1000x800x500x350x200x
RBS 5’UTR100ntGene
RangingCsrA Expression AnalyzingGeneExpressionthroughGFPReporter
GenesofInterest• glgC• maeB• aidB
Lacoperonpromoter Constitutivepromoter
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FlowCytometry
• Growovernightculture:• 5mlLB• Ampicillin• Kanamycin• Cellswithconstructs
Transferovernightcultureto10mlinErlenmeyerflask
WhenODreaches0.30,inducewithIPTG
IPTGintohalfofsamples
After3hours,analyzethroughflowcytometry
• GraphTitration• CalculateKd
TitrationMethod
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EstimatingtheBindingAffinity
-24.8
-23.1
-19.5
PredictedFreeEnergyofBinding(RTunits)
StrongerBinding
ExperimentalResults
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ImprovingtheModel
UTRStructure
RNASequence
BindingAffinityCalculator
EstimatedFreeEnergy
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FutureDirections
• AlterglgC loopsequence• EffectsofsequencemutationsonCsrA binding
• Testtitrationsystemonothergenes• pspA• sdhA• ythQ• mtlD
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Acknowledgements
• GrantGelderman• AbigailLeistra• LydiaM.Contreras,PhD• HowardSalis,PhD• OfficeoftheVicePresidentforResearch