南京大学-杨荣武分子生物学6课件

Settingupatransformation--howwillthecompetentcellsbetreated?Noplasmid(negativecontrol,nothingshouldgrowonthisplate)Supercoiledplasmidofaknownconcentration(todetermineefficiencyofcompetentcells,intransformants/microgram)VectorDNA(dephosphorylated?)ligatedwithoutinsertDNA(backgroundtransformants)VectorDNAligatedwithinsertDNA(desiredproducts)Exampleoutcomeofasuccessfultransformation:chemicallycompetentcellsNoDNA--Nocolonies2nanograms(10-9g,10-3micrograms)supercoiledplasmidDNA--500colonies(efficiencyofcells:2.5x105transformantspermicrogramDNA)Vectoralone--smallnumberofcoloniesVectorplusinsert--largernumberofcoloniesthanfor#3Identifyingrecombinantplasmid-containingcellsAlphacomplementation:mostwhitecoloniesrepresentpresenceofinsertDNAblockingfunctionalbetagalactosidaseIncreaseinnumberoftransformantsinpresenceofinsertvs.absenceofinsertVectortreatedwithalkalinephosphataseDirectionalcloning--preventingreligationofvectorSCREENcolonies/plasmidsforinserts,usuallybyPCRConfirmclonesbysequencingMobilizingDNA:vectorsforpropagationinE.coliPlasmidsBacteriophageM13LambdaSpecializedcloningvectorsexpressionvectorsandtagsvectorsforlargepiecesofDNA,e.g.CosmidsandBACsLambdaasacloningvectorInsertionalvectors(cloneintosinglerestrictionsite,canonlyincreasegenomesizeby5%(sizeofforeignDNAinsertdependsontheoriginalsizeofthephagevector,about5to11kb)Replacementvectors(removing“stuffer”),canclonelargerpiecesofDNA,8to24kb(sufficientformanyeukaryoticgenes)Cloninginlambdaphage--anoverviewLeftarmRightarm“Stuffer”Restrict,purifyrightandleftarms2)LigatewithforeignDNA3)“Package”ligationmixtureintophageheads4)PlatemixtureonE.coli,individualplaquesrepresentrecombinantclonesExamplesof“replacement”lambdavectorsFilamentousphages:M13Single-stranded,circulargenome,6.4kbCanclonepiecesofDNAupto6XtheM13genomesize(36kb)--butthelargertheDNA,thelessstablethecloneis…..UsefulforSequencingSite-directedmutagenesis(later)AnyothertechniquethatrequiressinglestrandedDNADrawback:foreignDNAcanbeunstable(slowsdownhostcellgrowth,sodeletionsconferaselectiveadvantage)M13doesn’tlysecells,butitdoesslowthemdownM13infectionsformplaques,buttheyare“turbid”“lawn”ofE.coliM13mp18:engineeredforalphacomplementationPhagemids:plasmid/M13hybrids

Plasmidscontainingbothplasmid(colE1)originandbacteriophageM13originofreplicationTorecoversingle-strandedversionoftheplasmid(forsequencing,e.g.),infecttransformed(male)strainwithahelperphage(M13KO7)Helperphagecannotproducesinglestrandedcopiesofitself,butprovidesreplicationmachineryforsingle-strandedcopiesofthephagemidDNAPhagemidsinglestrandedDNAispackagedandextrudedintosupernatant--canthenbeisolatedforsequencing,etc.UsesofBacteriophages:Lambda--large-ishDNAfragmentsforgenecloning(largeeukaryoticgenes)Excellentselectioncapability(stufferstuff)Clonelotsofprecisely-sizedDNAfragmentsforlibraryconstructionM13--single-strandedDNASequencingSite-directedmutagenesisEtc.

SpecializedvectorsforE.coliExpressionvectorsLargeDNAmolecules:Cosmids,PACs,andBACsExpressionvectorsForproductionofspecificRNAorproteinofinterestOptimizedfortranscription,translation,andpost-translationalhandlingTypicalexpressionvectorcloningsite:promoterMCStagstagsTranscriptionterminatorExpressionvectors:RNA:expressionoccursinvitro(purifiedplasmids)MakingmicroRNAsforRNAi:oneexampleHowtocontroltranscriptiondrivingRNA/proteinexpressioninvivo?T7RNApolymerasepromoters:T7RNApolymeraseundercontroloflacrepressor(inducedbyIPTG)LambdaPLpromoter,controlledbylambdarepressor(whichisregulatedbytrprepressor)pBADpromoter,controlledbythearaCproteininresponsetoarabinosepETvectors:proteinexpressionHelpertagsforproteinproductionandpurification

6/7histidinetag:interactsveryspecificallywithNi2+ions,whichcanbeimmobilizedoncolumnsorbeads

Biotincarboxylase:covalentlyattachestobiotin,biotinbindstostreptavidinwhichcanbeimmobilizedoncolumnsorbeads

Epitopes(e.g.c-myc)forspecificantibodiescanbeincludedastags--purifyonantibodycolumnTagscanbeengineeredtoberemovableUsingtagsinproteinpurificationhighaffinity,highspecificityAproteinpurificationscheme--removabletagCloninglargeDNAfragmentsCosmids:bacteriophagelambda-basedBacteriophageP1plasmidsBACs:Fplasmid-basedreplicontransferLambdacolE1P1P1FARStransfectiontransfectiontransfectionelectroporationelectroporationtransformationThisisaverygoodtabletobefamiliarwithWhyclonelargepiecesofDNA???Makelibraries:genomebrokenupintosmall,manageable,organizablepiecesEachrecombinantDNAfragmentfromtheligation--apieceofthegenomeHowmanyrecombinantDNAmoleculesarerequiredinalibrarytogetcompletecoverageofagenome?N=ln(1-p)ln(1-f)P=probabilityofgettingaspecificpieceofthegenome(1.0=100%)f=fractionalsizeofcloneDNArelativetogenomeN=numberofclonesneededN=ln(1-0.99)ln(1-)1.7x1043x10999%probabilityofhavingagivenDNAsequence17kbfragmentlibraryMammaliangenome:3x109basepairsN=8.1x105clonesrequiredCosmids:5kbplasmids,antibioticresistance,plasmidoriginofreplicationContainlambdacossitesrequiredforpackagingintolambdaphageheadsPackagingonlyoccurswith37-52kbfragments--selectionforlargefragmentsPackagedDNAisinsertedintocellsandthenreplicatesasaverylargeplasmidCloninginacosmidDesiredligationProducts--thesearepackagedCloninginacosmidInsteadoftransformation,desiredligationproductsarepackagedandthentransfectedintocellsSelectionforcolonies,notscreeningofplaques(notinfectious)Cosmids:aspecificcloningschemesplitSau3A:GATC5’overhang(compatiblewithBamHIstickyend)PreventsligationwithoutinsertPreventsmultiplefragmentsPhageP1vectors:cloningupto100kbDNAfragments85-100kbPhageP1vectors:cloningupto100kbDNAfragmentsEfficiencyofpackagingistypicallylow:thusitisnotgoodformakinglargegenomiclibrariesPhageP1vectors:cloningupto100kbDNAfragmentsPACs:likeP1vectorsbuttheDNAisnotpackaged(transferbyelectroporation)BACs:BacterialArtificialChromosomes

BasedontheFfactorofE.coli:--100kbplasmid,propagatesthroughconjugation--lowcopynumber(1-2copiespercell)--2genes(parAandparB):accuratepartitioningduringcelldivisionBACs:justhavepargenes,replicationori,cloningsites,selectablemarkerCanpropagateverylargepiecesofDNA:upto300kbRelativelyeasytomanipulate:moveintocellsbytransformation(electroporation)GeneralBACvectorreplicationselectionCloning,etc7kbo----Cloningstrategies----oMakingDNA“libraries”(fromgenomicDNA,mRNA“transcr