ObtainingbindingaffinitiesofCsrAtodifferentuntranslatedregions(UTRs)toimprovemodelforpredictionofCsrA interactions

AlexMoon-WalkerGrantGelderman andAbigailLeistra

Dr.LydiaM.ContrerasDepartmentofChemicalEngineering,TheUniversityofTexasatAustin

E.coli

GeneralFacts• Gramnegativerod• 4.6millionbasepairgenome• Partofgutflora

E.ColiintheWorldofMetabolicEngineering• Fastgrowth• Relativelysimplebiologicalsystem• AbilitytotakeupplasmidswithforeignDNA

https://c1.staticflickr.com/9/8723/16578744517_ed4293d3e7_b.jpg

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

CentralDogmaofBiology

DNARNA Protein

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.

TheCarbonStorageRegulatory(Csr)System

CsrA bindstocanonicalANGGAsequencesin5’UTRs

A U G C N

ANGGA

CsrA CsrA

CsrA bindstocanonicalANGGAsequencesin5’UTRs

A U G C N

ANGGA

CsrA CsrA

Controloftranslationalregulationsystemspresentsgreatopportunitiesformetabolicengineering

CsrA BindingPredictiveModel

UTRStructure

RNASequence

BindingAffinityCalculator

EstimatedFreeEnergy

CurrentProblem:Howaccurateisthemodel’sevaluationandpredictionofCsrAtargets?

Hypothesis

Estimatedbindingaffinityconstantsfrommodelcapturephysiologically-relevantCsrA-mRNAaffinities.

UsingGFPasreportertoanalyzechangesingeneexpression

InducedCsrA

Uninduced CsrA

DeoD genetarget

PlasmidConstructs

9000x5000x2000x1000x800x500x350x200x

RBS 5’UTR100ntGene

RangingCsrA Expression AnalyzingGeneExpressionthroughGFPReporter

GenesofInterest• glgC• maeB• aidB

Lacoperonpromoter Constitutivepromoter

FlowCytometry

• Growovernightculture:• 5mlLB• Ampicillin• Kanamycin• Cellswithconstructs

Transferovernightcultureto10mlinErlenmeyerflask

WhenODreaches0.30,inducewithIPTG

IPTGintohalfofsamples

After3hours,analyzethroughflowcytometry

• GraphTitration• CalculateKd

TitrationMethod

EstimatingtheBindingAffinity

-24.8

-23.1

-19.5

PredictedFreeEnergyofBinding(RTunits)

StrongerBinding

ExperimentalResults

ImprovingtheModel

UTRStructure

RNASequence

BindingAffinityCalculator

EstimatedFreeEnergy

FutureDirections

• AlterglgC loopsequence• EffectsofsequencemutationsonCsrA binding

• Testtitrationsystemonothergenes• pspA• sdhA• ythQ• mtlD

Acknowledgements

• GrantGelderman• AbigailLeistra• LydiaM.Contreras,PhD• HowardSalis,PhD• OfficeoftheVicePresidentforResearch

Questions?