GenomeAnalyzer

Introduction

Conclusions

Testing

IntegrationPlasmid

FeaturesofSynechocystis:•  Completelysequencedgenome•  Functionsasphotoautotrophor

heterotroph•  Carbonneutralmanufacturing

platform•  Diverseindustrialapplications:•  Fuels,plastics,foods,

pharmaceutics•  Capableofnaturaltransformation

TheplasmidpSB1A3_IntCwasdesignedtobecompatiblewithBioBrickformatsRFC10and23andwillmakecyanobacteriaamoreviableplatformforsyntheticbiologicalengineering.Thisplasmidenablesgenomemodificationvianaturaltransformationandhomologousrecombination.

AdvantagesofpSB1A3_IntC:•  Exchangeofrecombinationregionsforuseinmultiple

organismsormultiplelocationsinthesamegenome•  Insertionofgenecircuitsinanyregionofthegenome•  Abilitytoperformgeneknockouts•  Moreprecisecontrolofcopynumberthanwithplasmids•  Constructismaintainedingenomewithoutselection•  ConstructionofgeneconstructscanbeperformedinE.coli

Cyanobacteriaarerobustplatformsforengineering bioproducts.ThecyanobacteriumSynechocystissp.PCC6803isamodelorganisminmicrobiology.But,despitethemanypotentialusesandthepromiseofPCC6803ingeneticresearch,standardizedtechniquesformanipulatingandstudyingbacterialgeneticshavenotbeenadaptedtothisspecies.WiththedevelopmentofastandardizedtoolkitforPCC6803,thisspeciescanbecomeavaluableplatformforsyntheticbiologicalengineering.

Fig.1:Photobioreactor

PlasmidConstruction

pSB1A32157BP

pSB1A3servedasbasisforplasmidconstruction.

Fourrestrictionsitesintroducedusingsitedirectedmutagenesis.

XhoIBamHI NheI

NdeI NdeI

NheI

BamHI

XhoIHindIII

TworegionshomologoustoPCC6803Genomeclonedintonewsites.HindIIIsiteinsertedduringcloning.

BBaK3900002157BP Intermediate BBa_K390200

Resistancegeneclonedintointegrationregion.

Recombinationregionscanbeexchangedtomeetneeds.

XhoI

HindIII

BamHI

NheI

NdeI

DesiredgeneconstructinsertedintoBioBrickstandardcloningsites.

Synthetic Bio-Manufacturing Center

CyanoBricksDevelopingCyanobacteriaasaBiologicalEngineeringPlatformC.Tramp,A.Tejeda,A.Hatch,C.Peterson,E.Monzon,S.Shen,B.Henrie,R.C.Sims,H.S.Hinton,C.Miller

Fig.2:HomologousRecombinationMechanismFig.6:BioBrickpartsgeneratedinthisstudy.Thesepartswerecombinedtomakecompositeparts.

All Synechocystis promoterswereconstructedwiththeirnativeribosomebindingsites(RBS).Asub‐groupofthesepromoterswerealsopairedwiththeconsensusRBSderivedfromthegenomeanalyzersoftware.AllpartswereconstructedinE.coliby

BioBricksaddingGFP(cycle3mutant)andadoubleterminatortothepromoterandRBSfromPCC6803.

Fig.8:GFPfluorescenceoftestedBiobricksinE.coli.BecausepromoterpsaABhadthemostconsistentfluorescencelevelsthroughouttestingperiod,allotherreadingswerenormalizedrelativetopsaAB

ThegoalofthisprojectwastodevelopandcharacterizeaplatformforengineeringbioproductsinSynechocystissp.PCC6803.WeaccomplishedthisgoalbyisolatingandtestingpromotersandRBSfromvariousnativegenes.AnintegrationplasmidwasdevelopedtoaddconstructsdirectlyintotheSynechocystisgenome.Projectachievementsinclude:

Fig.3:(left)ScreenshotsfromtheGenomeAnalyzerillustratingmanyprogramcapabilities.Fig.4:(right)CodonUsageAnalysisofSynechocystisGenome.Valuescalculatedusingeverygeneinthegenome,togiveanaccuratereflectionoftRNApreference.Fig.5:(bottom)RibosomebindingsiteconsensussequenceasdeterminedbyGenomeAnalyzerprogramanalyzingtheupstreamregionofeverygeneinthegenome.Colorsscaleindicateslevelofconservation.TheconsensusRBSsequencefromE.coliisshownforcomparison.

FunctionalityofBioBrickscontainingpromoterandRBSfromnativegeneswasdeterminedusingaGFPreporter.BeforeinsertionintoPCC6803,constructsassembledinpSB1C3weretestedinE.coli.ComparisonofPCC6803promotersandRBStotheconsensuspromoterandRBSofE.colirevealssomedegreeofhomology(Fig.7).

FluorescencelevelsofE.colicontainingconstructsweremeasuredspectrophotometricallythroughoutthegrowthperiod.VariablelevelsofexpressionsuggestfunctionalityofsomeofthepromotersinE.coli.FutureworkwillfocusoncharacterizingexpressionlevelsofthesecompositepartsinSynechocystissp.PCC6803.

Fig.7:SequencealignmentofpromotersandRBSwithE.coliconsensus

FutureworkwillfocusontestingtheintegrationplasmidandfurthercharacterizationofpromoterfunctioninSynechocystissp.PCC6803.

Agenomeanalysisprogramwasdevelopedtoaidwiththedesignofthenewintegrationplatform.ThisprogramcandownloadtheGenBankgenomefilewhichitusestocompileasearchabledatabaseofgeneinformation.Thisdatabasecontainsinformationongenesequences,names,functions,locations,andadditionalnotes.ItalsoallowedustoanalyzetheconservationofeachbaseintheRBS,thecodonusageprofile,andthepotentialpromoterslocatedintheregionsupstreamofthegenes.

•  Constructionofagenomeanalyzerprogram•  Assemblyandsumbissionof43BioBricks•  Constructionofanintegrationplasmid