分子實驗報告2010030038生05陶欣

Thesummerexperiment——theMolecularPartBio05陶欣2010030038Group8-2ClassBExperimentpartner:劉子源金雨2012/8/24-2012/8/28ObjectivesTheoverallobjectiveofthissummerall-aroundexperimentistoimproveourexperimentaloperationskillsandproblem-designingskills,tobuildupourgoodhabitsinworkingandstudy,tolearnhowtocooperatewithourpartners,andtoobtainacertainlevelofstandardwritingabilitythroughtheprocessoftheexpression,purificationandidentificationoftheHSP16.3protein.BackgroundandPrinciplesSmallheatshockprotein(sHSP)Inresponsetoenvironmentalstresses,cellsincreasetheexpressionofoneclassofimportantproteinscalledsmallheatshockproteins(sHSP),whichendowcellswiththermo-toleranceandarebelievedtobeinvolvedintheinhibitionofapoptosis,organizationofthecytoskeleton,establishingtherefractivepropertiesoftheeyslens,andstabilizationofRNA.ProteinsofsHSPfamilyhavevariousbiologicalfunctions,butallofthemsharetheATPindependentchaperoneactivity,preventingproteinaggregationinstress.Structurestudiesindicatethattheyusuallyfunctionbyassemblingintocertainoligo-structure.HSP16.3HSP16.3isanimportantsHSPfromMycobacteriumTuberculosi,itismarkedlyexpressedduringthestationaryphaseandbecomesamainproteininthecell.HSP16.3isanimmunodominantantigenandthemajormembraneproteinofM.tuberculosis.PreviousstudiesshowedthatHSP16.3canmakethecellstructurestableandpreventstationaryM.tuberculosisfromautolysing.GeneknockoutstudiesindicatethatHSP16.3isrequiredforM.tuberculosistogrowinmacrophages.HSP16.3consistsof144aminoacidsanditsmolecularweightis16277Da.Itisaα-crystallin-relatedsHSPwiththeconservedα-crystallindomaininitsC-terminalpart(D/N-G-V-L-T-T/V-X-V/A).ExperimentsinvitrorevealedthatHSP16.3cansuppressthethermalaggregationofcitratesynthaseat39.5?C,withoutconsumptionofATP.Itusuallyassemblesintoatriangulartrimer-of-trimerssturtureinvitro.pET-28aplasmidFigure.1thestructureofpET-28aplasmidIntroductionofPCRThepolymerasechainreaction(PCR)isatechniqueforamplifyingDNAsequencesinvitro.ThismethodtakesadvantageofthermallystableDNApolymeraseandcanproducenumerouscopies(about268,435,456)fromasingletemplateDNAmoleculethroughtensofrepeatedcyclesoftemplatedenaturation,primerannealing,andDNAsynthesis,butitisextremelysensitivetotracecontaminationofunwantedtemplateDNAinthereactionsolution.Thismethodisalsoimportantinbiotechnology,forensicidentification,medicine,andgeneticresearch.PCRwasinventedbyKaryMullisandhiscolleaguesin1985.ThediscoveryofTaqDNApolymerase,thermallystableenzymeisolatedbyChienetal.in1976,madethePCRautomationpossible.In1987,KaryMullisetal.accomplishedthePCRautomationsystemwhichmadePCRpractical.KaryMulliswasawardedthe1993NobelPrizeinChemistryforinventingPCR.PCRhasplayedamajorroleintheInternationalHumanGenomeProject,whichhasmajorhealthandantiagingimplications.Thetechniquehasalsobecomeinvaluableinbiotechnology,forensicidentification,medicine,diseasediagnosis,forensic-scienceanalysisinconvictingtheguiltyandfreeingthefalselyaccused,andthestudyofDNAfromancientorfossiltissues.TheprincipleissimilartoDNAdenaturationandrenaturationathightemperature(93-95℃),thetargetdouble-strandDNAcanbeseparatedintosingle-strandDNA.Atlowtemperature(37-65℃),twoartificialoligonucleotideswillannealedtothecomplementarysequenceinthetemplateformingpartialdoublestrand.At72℃,theTaqpolymerasesynthesizesnewstrandbyextendingtheprimersalongthedirectionfrom5’to3’.Thenumberofsequencesbetweentheprimersisdoubledafterthiscycle.ThecyclecanberepeatedasthenewlysynthesizedDNAstrandscanserveastemplatesinthenextcycle.So,theamountoftargetsequencedramaticallyincreases.Theamplificationcoefficientcanbeabove109theoreticallyafter25-30cyclesor106-107practically.GelelectrophoresisBymeltingandthenrefreezingagarose,itcanformakindofsolidbasewithtinyholes,whichiscalledagarosegel.DNAmoleculescanbeinjectedintoonesideofthegel.Then,whentheDNAmoleculesaresubjectedtoanelectricfieldwiththepresenceofappropriatebuffer,theDNAmoleculeswillmovetowardtheanodeindifferentvelocitiesaccordingtothedistinctivestructureandmolecularmassofDNA.(SeeFig.2)Figure.2GelElectrophoresisSeveralfactorsarecrucialindeterminingthemobilityshiftoftheDNAmolecules,suchasthesizeandstructureoftheDNA,themolecularmassoftheDNA,pressure,thebuffer,etc.CompetentcellsandtransformationNaturally,bacterialcellsarenoteasytoallowforeignDNAtopasstheircellwalls.However,whentheyaretreatedwithsomereagents,suchasCaCl2,theircellwallswillbealteredwhichmakestheforeignDNAenterthecellseasily.Suchcellsarecalled“competentcells”.TheprocessofintroducingforeignDNAintothecompetentcellsiscalled“transformation”.Thetransformationisafundamentaltechniqueingeneengineering.Throughthistechnique,theforeignDNAcanbereplicatedandexpressedinthecompetentcellswhichmakesthehostcellsgainnewgeneticphenotype.Thecompetentcellsusedinthetransformationaregenerallyrestriction/modification-deficientmutantswhichhavenorestrictionandmethylationenzymes.Threemajorpartoftheexperiment—molecularbiologypart,microbiologypart,andbiochemistrypart.Hereinthemolecularbiologypart,theHSP16.3gene(togetherwithaKanamycinresistancegene)isamplifiedbyPCRandclonedintotheplasmidpET-28a,andthenweusetherecombinantplasmidtotransformE.Coli.BL21strain.Materials,reagentsandequipmentsElectrophoresisMaterialsDNAsampleNEB1KbDNALadder(dilutedto50ng/μl)aragoseReagents:5×TAEbuffer10×loadingbufferEBEquipments:ElectrophoresisinstrumentElectrophoresistroughPipette&tipsMicrowaveovenUltraviolettransmissionInstrumentGelImagingSystemPCRMaterials:TemplateDNA(containingtheKanamycinresistancegene,dilutedbeforehand)Upstreamanddownstreamprimers(dilutedto25μMbeforehand)UpstreamprimerhspF：5'-CAGCCACCACCCTTCCCGTTCA-3'DownstreamprimerhspR：5'-CCGGTTGGTGGACCGGATCTGAA-3'Reagents:10×PCRBuffer(Mg2+plus)dNTPMixture(2.5mMeach)TaKaRaTaqPolymerase5U/μlEquipments:PipettesandtipsPCRtubesPCRmachinePurificationofthePCRproductsSmallscaleDNAfragmentpurificationKit(BioDevCorp):BindingbufferCon.WashsolutionAbsorbanceSpinColumnWasteLiquidCollectionTubeRestrictionenzymedigestionofPCRproductandthevectorMaterials:PCRproductpET-28avectorReagents:BamHI(15U/ul)XholI(10U/ul)EnzymedigestionbufferEquipents:WaterbathPipetteandtipsEPtubesIceboxPurificationofthedigestedPCRproductMaterials:EnzymedigestedPCRproductReagents:100%ethanol70%ethanoldistilledwater3MNaAcEquipents:RefrigeratorCentrifugeEPtubesPipetteandtipsPurificationofthedigestedvectorRecoveryfromGel-SmallScaleDNAFragmentPurificationKit(BioDev):GeldissolvingsolutionCon.WashSolutionTypeBAbsorbanceSpinColumn/WasteLiquidCollectionTubeLigationMaterials:DigestedandpurifiedPCRproductDigestedandpurifiedpET-28avector(Kanamycin,lacZ),5.3kbReagents:DNALigationKitVer2.1(TaKaRa)Equipents:IceboxCompetentE.colicellpreparationMaterials:E.coliBL21strainReagents:LBculturemedium(solidandliquid)AmpcontainingLBsolidculturemedium0.05mol/LCaCl20.05mol/LCaCl2containing15%glycerolEquipents:Pipette&tipsEPtubesShakerflaskIncubatorCentrifugeRefrigeratorWaterbathIceboxCleandeskSpetrophotometerTransformationMaterials:Competentcells:E.coliBL21strainRecombinantplasmidReagents:LBculturemedium(solidandliquid)KanamycinsolutionEquipents:ShakerflaskCentrifugeWaterbathIncubatorCleandeskIceboxPipette&tipsEPtubesSelectionplateSteriletoothpickIsolationoftheplasmidMaterials:TransformedandselectedE.colicellsReagentsandequipments:Smallscaleplasmidrapidextractionkit(BioDevCorp.)EnzymaticdigestionandelectrophoresistoidentifythepositiverecombinantplasmidMaterials:IsolatedplasmidfromthetransformedandselectedE.coliDH5αcellsNEB1KbDNALadder(dilutedto50ng/μl)aragoseReagents:10XBufferK,BamHI,XhoI(TaKaRaCorp.)5×TAEbuffer10×loadingbufferEBEquipents:ElectrophoresisinstrumentElectrophoresistroughPipette&tipsMicrowaveovenUltraviolettransmissionInstrumentGelImagingSystem=4\*ROMANIVProceduresPreparationoftargetHSP16.3DNAThe50μlPCRsystemisasfollows:Table.1PCRreactionsystemsetMateralorreagentVolumeandconcentrationddH2O37.5μl10×PCRBuffer(Mg2+plus)5μldNTPMixture4μlTemplateDNA2μlalUpstreamprimer0.5μlDownstreamprimer0.5μlTemplateDNA2μlTaKaRaTaqpolymerase0.5μlTotalvolume50μl-32cycles:95℃for5min,95℃denaturationfor40s,55℃annealingfor30s,72℃elongationfor40s.-Afterthelastcycle,72℃for7min,heatpreservationunder4℃.PurificationofthePCRproduct1）AdjustthevolumeofthePCRsampleto50μlwithwaterifnecessary.Add8volumesofBindingBufferto1volumeofthePCRsampleandmix.(Note:Forastandardpurification,add400ulofbindingbufferto50ulPCRsample)2）Placethespincolumninaprovided2mlcollectiontube.3）Applythesampletothecolumnandcentrifugeat12,000rpmfor30s.4）Discardflow-through.Placethecolumnbackintothesametube.(Note:Collectiontubesarere-usedtoreduceplasticwaste.)5）Add500μlWashingBuffertothecolumnandcentrifugeat12,000rpmfor30s.(Note：Add3volumeofethanol(100%)to1volumeofConcentratedWashingBufferbeforeuse.)6）Repeatstepaboveforonetime.7）Discardflow-throughandplacethecolumnbackinthesametube.Centrifugethecolumnforanadditional2min.(IMPORTANT:ResidualwashingBuffershouldbecompletelyremovedcarefullywithpipettetipifnecessary.)8） Placethecolumninaclean1.5mlmicrocentrifugetube.9）Add40mlH2Otothecenterofthecolumnmembrane,letthecolumnstandfor1min,andthencentrifugeat12,000rpmfor30s.10）Takeoutthemicrocentrifugetube.Reservetheflow-throughinatube. ElectrophoresisofthepurifiedPCRproduct.Inthisprocedure,thepurposeistoidentifywhetherthereisPCRproduct.Thegelisprepared.Mixthe3μlPCRproductwiththe2μlloadingbuffer.Andthenloadthebuffertothewell.Runthegelunder100V.Observetheresultoftheelectrophoresis.EnzymedigestionofthePCRproduct.The20μldoubledigestionsystemisdesignedasfollow.Table.2restrictionenzymedigestionofthepurifiedPCRproductMateralorreagentvolumePurifiedPCRproduct6lBamHI1lXholI1l10×Kbuffer2lddH2O10lTotalvolume20l-Waterbathat37℃for2hours.PurificationofthedigestedPCRproduct.Add50lethanolandNaAc(pH=5.2)tothedigestedPCRproduct,-20℃intherefrigeratorfor20min.4℃,12000rpm,centrifugatefor8min,discardthesupernatant.Add500l70%ethanoltowashtheprecipitate,12,000rpmcentrifugateatroomtemperaturefor3min.DrytheDNAprecipitateatroomtemperaturefor15minorso,add40μlH2OtoresolvethedigestedPCRproduct.2.PreparationofthepET-28aplasmidRestrictionenzymedigestionofthevectors.The50ldoubledigestion(BamHI/XholI)systemofthepET-28avectorisshownasfollows:Table.3restrictionenzymedigestionofpET-28aMaterialorreagentvolumepET-28avector20lBamHI2lXhoII3l10×Kbuffer5lddH2O20lTotalvolume50l-37℃waterbathfor5-6hrs.PurificationofthedigestedvectorRunthedigestedvectoronthegel,cutofftheband,keepingthegelsmallest.(tryyourbesttoremovetheemptygelwhichdoesn’tcontainthevector,ifyoukeeptoomuchemptygel,younotonlywastethegelliquid,butalsodecreasetherecoveryefficiencyofdigestedvector.)Addsolaccordingtotheprincipleof1:5ratioofgelandsol,meltingthegelat55℃-65℃,shakingtheEPoccasionally.Addthemeltedgelintospincolumn,centrifugeitat12000rpmfor30s,discardtheflow-through.Add500μlWashingBuffertothecolumnandcentrifugeat12,000rpmfor30s.Repeatthestepaboveforonetime.Discardflow-throughandplacethecolumnbackinthesametube.Centrifugethecolumnforanadditional2min.(IMPORTANT:ResidualwashingBuffershouldbecompletelyremovedcarefullywithpipettetipifnecessary.)PlacethecolumninacleanEPtube.Add10mlH2Otothecenterofthecolumnmembrane,letthecolumnstandfor1min,andthencentrifugeat12,000rpmfor2min.Reservetheflow-through.3.PreparationoftherecombinantplasmidsElectrophoresisofthevectorandthetargetfragmentPlace1μlloadingbufferand3μlthetargetDNAfragmentsolutiononapieceofsuperhydrophobicpaperinadropletandmixitupwithapippet.Addallthe4μlsolutionintoawellintheagarosegel,runthegelunder100V.Place2μlloadingbufferand3μlthedigestedvectorsolutiononapieceofsuperhydrophobicpaperinadropletandmixitupwithapipette.Addallthe4μlsolutionintoawellintheagarosegel,runthegelunder100V.Observetheresultoftheelectrophoresis.LinkageofthevectorandtargetfragmentMixthevectorandtargetDNAfragmenttomake7μlDNAsolution.(weusethevectorfromtheteacher,theamountis3μl,thusthefragmentis4μl)Add7μlSolutionItotheDNAsolution(ontheice)andmixup.Reactat160Cfor1hour.Addthissolutionto100mlE.coliDH5αcompetentcells.(onice.)4.TransformationandcultivationTransformationTake100mlE.coliDH5αcompetentcellsfrom-70fridge.Putontheiceboximmediatelyafterthawing.Addtherecombinedplasmidtothetube,mixitgentlyandputintheicefor30min.Heatshockbytransferringthetubestoawaterbathof42℃for90seconds.Immediatelyreturnthetubetotheicebath.Keeponicefor2~3minutes.Add0.5mlofLB(withnoantibioticsadded)intoeachtube.Incubatethetubesfor45minutesat37℃toallowthecellstoexpresstheirantibioticgeneproduct.Centrifugethetube2minat4000rpm.Discardthesupernatantuntilthere200μlleft.Mixitandspreadthe200μloftheresultingsolutionsonLBplates(withkanamycin).AftercompleteabsorptionofliquidLB,upsidedowntheplatesandincubatetheplatesat37℃overnight.CultivationPickthecolonywithaseptictoothpick;loadthecloneson3mlLBliquidculturemedium(kanamycin50μg/ml)at37℃on180rpmshakingtablefor12h~16h.5.IdentificationandretransformationIsolationofplasmidsLoad3mlmediumintoEppendorftube,10000r/mincentrifuge1min，giveupsupernatant，add100μlsolutionI(GET)，blendfor3-5min.Add200μl0.2mol/LNaOH+1％SDS（solutionII），mixthesolutionfor6-7times,keepthetubeinicefor5min.Add150μlpotassiumacetate(solutionIII),mixthesolutionfor6-7times,keepthetubeinicefor5min.Subjectthetubetohigh-speedcentrifuge10000r/mincentrifugefor7min,putthesupernatantinanewtube,add1ml100％ethanol，12000r/mincentrifuge5min，giveupsupernatant.Washthesedimentationwith0.5ml70％ethanol，12000r/mincentrifuge5min，giveupsupernatant，putthemonbibulouspaper，desiccateinlab.Add50μlwater,keepfor1~2min,centrifugeat12000rpmfor1min.DigestionandidentificationTable.4digestionoftheplasmidMaterialorreagentvolume10XBufferK2μlBamHI1μlXhoI1μlPlasmid10μlddwater6μlTotalvolume20μl-WaterbathandtherunelectrophoresisgelTransformationoftherecombinantDNAtoBL21Take100mlE.coliBL21competentcellsfrom-70fridge.Putontheiceboximmediatelyafterthawing.Addtherecombinedplasmidtothetube,mixitgentlyandputintheicefor30min.Heatshockbytransferringthetubestoawaterbathof42℃for90seconds.Immediatelyreturnthetubetotheicebath.Keeponicefor2~3minutes.Add1μlofLB(withnoantibioticsadded)intoeachtube.Incubatethetubesfor45minutesat37℃toallowthecellstoexpresstheirantibioticgeneproduct.spreadthe200μloftheresultingsolutionsonLBplates(withkanamycin).AftercompleteabsorptionofliquidLB,upsidedowntheplatesandincubatetheplatesat37℃overnight.PreservethepositivecloneTakeacloneofE.Coli.BL21to500μlLBmediumcontaining50μg/mlkanamycin.Incubatetheculture4~5hoursat37degree.Add500μlglyceroltothetube,storeat-20℃.ResultsanddiscussionElectrophoresisofthePCRproduct(afterpurification)ResultBeforethepurificationofthePCRproduct,werunanelectrophoresisofthePCRproductandgettheresultasfollowsinFig.3.12345678910111213141516171234567891011121314151617Figure.3theelectrophoresisresultofPCRproductandthestandardmarker(lane16)Oursampleisaddedinlane8,therightgel.DiscussionAswehaveknownfromreference,thePCRproductofHSP16.3geneisabout430bplong.Butmostofthesampleinourclassisaround500bp.Wecancalculatedthatoursampleisabout500bpreferringtotheNEBDNAladder.ButwecanstillconsiderthatwegettherightPCRsegmentaswehaveexpected.However,thebandinthelargegelisnotasbrightasthelittle’s.Andthepossiblereasonscomefromtwoaspectsbelow:Somemistakesaremadeinthestainingstepsothatthebandsareallnotsobright.InthestepofpurifyingPCRproduct,wemaynotremovethewashingsolutionclearlysothatsomeethanolremaininthevessel,resultinginthelowrecoveryefficiency.ElectrophoresisofthedigestedvectorandtargetfragmentResultwehavetocomparethebrightnessofthevectorwiththatoftheDNAfragmenttodeterminethevolumeofthetwosamplesintheligationstep.Besides,weaisohavetoexaminewhetherthevectorisfullycutbyenzymeandcondensethedigestedvector.Werunanelectrophoresisofthedigestedvectorandgettheresultasfollows.910111213141516191011121314151612345678Figure.4TheelectrophoresisresultofdigestedvectorandthestandardmarkerLane6isthestandardmarkerandLane4ismygroup’svector.DiscussionOurvectorisonlane4andDNAfragmentonlane7.Butwecan’tseeanythinginlane4andlane7.Thepossiblereasonsmaybethatthedigestiontimeisnotenoughandthefragmentsarenotenoughtosee.Sowehavetouseothergroup’ssampleswiththeirvectoronlane6andDNAfragmentonlane3.Aswehaveknownfromreference,thelengthofthevectorisabout5.3KbandtheDNAfragmentisabout430bp.WeconsiderthattheDNAfragmentis4timesbrighterthanthevector.Thus,tomakesuretheDNAfragment:thevectorisfrom10:1to3:1,weadd1μlDNAfragmentand6μlvector.Thecalculationproceduresareasfollow:V1+V2=7V1/V2=5300/(3*430N)(Nisthebrightnessratio,V1representsvolumeofvector,V2representsvolumeoftargetDNAfragment)CultivationofthebacteriaAfterovernightcultivation,wegetmanycoloniesinthecultureplateandwedidn’tseeanyother