分子大实验-乃哥麦提-2011012325VIP免费

ComprehensiveexperimentsofBiochemistryandMolecularBiologyMolecularPart生13杨林枫2011012326同组人员：乃哥麦提褚培睿I.Objectives1.Learntodesignanentiremolecularbiologyexperimenttogetasystemwhichcanexpressprotein.2.Reviewthemolecularbiologyexperimenttechnologywelearnedbefore.3.PreparethesamplesforpartIIandpartIII.4.Organizingeachassaytogetfullyuseofthetime.5.Corporatewithothers.II.Background1.HSP16.3HSP16.3,asmallHeatShockProteinfromM.tuberculosis,consistsof144aminoacidsandhasamolecularweightof16277Da.ItissignificantlyexpressedwhenM.tuberculosischangesfromlogarithmphasetostablephaseandbecomesmainproteininthecell.HSP16.3isalsoanantigen,importanttargetsiteofTandBcellinimmuneresponse.ExperimentsinvitroindicatethatHSP16.3hasachaperoneactivity,anditcaninhibitthermalaggregationofcitricacidsynthetaseat39.5withoutconsumptionofATP.Butitcannotprotectcitricacidsynthetaseactivity,whichindicatesthatHSP16.3interactswithpartiallyunfoldingcitricacidsynthetaseandrefoldingofproteininvivomayneedotherproteins.GelchromatographycanseparateHSP16.3-citricacidsynthetasecomplex,andSDS-PAGEcanidentifythem.HSP16.3mayexposeitshydrophobicsurfacetocarryoutitschaperoneactivity.Withmildtemperatureandmutageninlowconcentration,forexample,undertheconditionof0.05MGuanidiniumchloride,0.3Mureaand30,HSP16.3willexposeitshydrophobicsurfaceandelevateitschaperoneactivity.HSP16.3isamemberof-crystallineassociatedsmallHeatShockProtein(sHSP)family,withthetypicalconservedCTD:D/N-G-V-L-T-T/V-X-V/A.sHSPhavemanydifferentbiologicalfunctions.Theyallhavechaperoneactivity,interactingwithdenaturedproteinsubstrateevenwithoutATP.Researchshowsthattheyoftenformcertainoligomerstructuretocarryoutitsfunction,forexample,M.JanneschiiHSP16.5forms24-oligomericstructureandTriticumaestivumHSP16.9forms12-oligomericstructure.HSP16.3isoneoftheseveralsHSPfromprokaryotesthathavebeenfound.Thefunctionmechanismofitisnotclearyet.2.PET-28aplasmidLibraryID:G0306Name:pET-28a(+)Type:BacterialResistance:KanamycinConditionofculture:GrowinstandardE.coliat37℃Figure1.pET-28aplasmidTherearemodulessuchaslacoperator,whichfacilitatesartificialinductionofgeneexpression,Histag,whichfacilitatesproteinpurification,andMCSinthevector.3.BackgroundaboutPCRThepolymerasechainreaction(PCR)isatechniqueforamplifyingDNAsequencesinvitro.ThismethodtakesadvantageofthermallystableDNApolymeraseandcanproducenumerouscopies(about268,435,456)ofDNAsfromasingletemplateDNAmoleculethroughtensofrepeatedcyclesoftemplatedenaturation,primerannealingandDNAsynthesis.ItisextremelysensitivetotracecontaminationofunwantedtemplateDNAinthereactionsolution.Thismethodisalsoimportantinbiotechnology,forensicidentification,medicineandgeneticresearch.PCRwasinventedbyKaryMullisandhiscolleaguesin1985.ThediscoveryofTaqDNApolymerase,thermallystableenzymeisolatedbyChienetal.in1976,madethePCRautomationpossible.In1987,KaryMullisetal.accomplishedthePCRautomationsystemwhichmadePCRpractical.KaryMulliswasawardedthe1993NobelPrizeinChemistryforinventingPCR.PCRhasplayedamajorroleintheHumanGenomeProject.Thetechniquehasalsobecomeinvaluableinbiotechnology,medicine,diseasediagnosis,forensic-scienceanalysisinconvictingtheguiltyandfreeingthefalselyaccused,andthestudyofDNAfromancientorfossiltissues.Figure2.Thepolymerasechainreaction(PCR)4.GelElectrophoresisAftermeltedinhightemperature,theagarosesolutionwillformsolidwithcertain...