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Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual
PT3247-1 (PR742219)Published 3 April 2007
Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3247-1 2 Version No. PR742219
I. Introduction 4 II. Overview:AYeastTwo-HybridScreen 8
III. ListsofComponents 9
IV. AdditionalMaterialsRequired 10
V. YeastStrains&Phenotypes 11 A. YeastHostStrains 11 B. Phenotypes 11
VI. ControlVectors 13 A. PositiveControls 13 B. NegativeControl 13
VII. Matchmaker™GAL4cDNA&GenomicLibraries 14 A. LibraryConstruction 14 B. LibraryQualityControlInformation 15
VIII. PreparingforaTwo-HybridScreen 16 A. ConstructFusionGenes 16 B. ObtainorConstructandADFusionLibrary 16 C. VerifythatConstructsDoNotActivateReporterGenes 17 D. VerifyProteinExpression 17
IX. LibraryTransformation&ScreeningProtocols 18 A. TransformationScales 18 B. YeastTransformationProtocols 19 C. PlatingandScreeningTransformationMixtures 21 D. Calculations 23
X. Analysis&VerificationofPutativePositiveClones 24 A. RetestPhenotypes 24 B. IsolatePlasmidDNAfromYeast 24 C. SortColoniestoEliminateDuplicates 24 D. RescueAD/libraryPlasmidsviatransformationofE.coli 24 E. RetestProteinInteractionsinYeast 25 F. InvitroAnalysis 26 G. SequencingAD/LibraryInserts 27 H. InvivoAnalysis 27 I. AdditionalTwo-HybridTests 27
XI. TroubleshootingGuide 28
XII. References 30
XIII. RelatedProducts 32
AppendixA:Media&SolutionRecipes 33
AppendixB:LibraryTitering 34
AppendixC:PlasmidLibraryAmplification 35
TableofContents
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Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual
ListofTables
TableI. ListofAbbreviations 7TableII. MatchmakerYeastStrainGenotypes 11TableIII. MatchmakerYeastStrainPhenotypes 11TableIV. MatchmakerTwo-HybridSystem3Vectors 13TableV. ComparisonofTwo-HybridTransformationMethods 18TableVI. SetupforaTwo-HybridLibraryScreen 21
ListofFigures
Figure1. OverviewofMatchmakerTwo-HybridSystem3advancesandscreeningprocess 4Figure2. Thetwo-hybridprinciple 5Figure3. ReporterconstructsinyeaststrainsAH109andY187 5Figure4. Overviewofperformingayeasttwo-hybridscreen 8Figure5. ScreeninganADfusionlibraryusingstrainAH109 22Figure6. Strategiesforanalyzingandverifyingputativepositiveclones 25Figure7. Yeastmatingtoverifyproteininteractions 26
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Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3247-1 4 Version No. PR742219
I. Introduction
Principleofthetwo-hybridassay
InMatchmakerSystem3abaitgeneisexpressedasafusiontotheGAL4DNA-bindingdomain(DNA-BD),whileanothergeneorcDNAisexpressedasafusiontotheGAL4activationdomain(AD;Fields&Song,1989;Chienet al.,1991).Whenbaitandlibraryfusionproteinsinteract,theDNA-BDandADarebroughtintoproximity,thusactivatingtranscriptionoffourreportergenes(Figure2).Thistechnologycanbeusedtoidentifynovelproteininteractions,confirmsuspectedinteractions,anddefineinteractingdomains.Moreover,two-hybridtechnologyprovidesimmediateaccesstothegenesencodingtheinteractingproteins.
Sensitivein vivoassay
Yeasttwo-hybridsystemsprovideasensitivemethodfordetectingrelativelyweakandtransientproteininteractions.Suchinteractionsmaynotbebiochemicallydetectable,butmaybecriticalforproperfunctioningofcomplexbiologicalsystems(Guarente,1993;Estojaket al.,1995).Thesensitivity of Matchmaker GAL4Two-Hybrid System 3 is primarily attributable to high-foldamplificationofpositive signals in vivo (i.e., transcriptional, translational, andenzymatic). Inaddition,becausethetwo-hybridassayisperformedin vivo,theproteinsaremorelikelytobeintheirnativeconformations,whichmayleadtoincreasedsensitivityandaccuracyofdetection.
MatchmakerTwo-HybridSystem3isanadvancedGAL4-basedtwo-hybridsystemthatprovidesatranscriptionalassayfordetectingproteininteractionsin vivoinyeast.Youcanusethissystemtoscreenalibraryfornovelproteinsthatinteractwithaknownbaitprotein,ortotesttwopreviouslyclonedproteinsforaninteraction.MatchmakerTwo-HybridSystem3incorporatesmanyfeaturesthatreducetheincidenceoffalsepositiveresultsandallowyoutoquicklyidentifyandconfirmproteininteractions.KeyfeaturesofSystem3aredetailedinFigure1.
Figure1.OverviewofMatchmaker™System3advancesandscreeningprocess.
Library screening
α-gal or β-gal assay
Verification oftrue positives
Ade+, His+
Colonies
Strain AH109
• Virtually eliminates false positives
• Allows stringency of selection to be varied
• Use simple α-gal or β-gal assay
Improved vectors
• Increased transformation efficiency
• High-level protein expression
• c-Myc and HA tags facilitate detection of fusion proteins
• Distinct bacterial selection markers AD—Ampr DNA-BD—Kanr
• High copy vectors • T7 promoter allows in vitro transcription and translation
Matchmaker™Co-IP Kit
Epitope-TaggedExpression Vectors
or MammalianMatchmaker™ Kit
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I. Introductioncontinued
Figure2.Thetwo-hybridprinciple.TheDNA-BDisaminoacids1–147oftheyeastGAL4protein,whichbindstotheGALUASupstreamofthereportergenes.TheADisaminoacids768–881oftheGAL4proteinandfunctionsasatranscriptionalactivator.
Thesensitivityofthetwo-hybridassaymeansthatitcanbeusedtopinpointspecificresiduescriticalforproteininteractionsandtoevaluateproteinvariantsfortherelativestrengthoftheirinteractions (Yang, et al.,1995).ThebindingdatareportedbyYanget al. (ibid.) lead themtosuggestthatproteininteractionswithdissociationconstants(Kd)above~70µMcanbedetectedusingaGAL4-basedtwo-hybridassay.
Newyeaststrainreducesfalsepositives
System3 features theyeast strainAH109,whichvirtuallyeliminates falsepositivesbyusingthreereporters—ADE2,HIS3,andMEL1 (or lacZ)—underthecontrolofdistinctGAL4upstreamactivatingsequences(UASs)andTATAboxes(Figure3).ThesepromotersyieldstrongandspecificresponsestoGAL4.Asaresult,twomajorclassesoffalsepositivesareeliminated:thosethatinteractdirectlywiththesequencesflankingtheGAL4bindingsiteandthosethatinteractwithtranscriptionfactorsboundtospecificTATAboxes.
TheADE2 reporteraloneprovidesstrongnutritionalselection:theoptionofusingHIS3selectionreducesthe incidenceof falsepositivesandallowsyoutocontrol thestringencyofselection(Jameset al.,1996).Furthermore,youhavetheoptionofusingeitherMEL1orlacZ,whichencodeα-galactosidaseandβ-galactosidase,respectively.MEL1isanendogenousgenefoundinseveralyeaststrains.Becauseα-galactosidaseisasecretedenzyme,itcanbeassayeddirectlyonX-α-Gal(Cat.No.630407)indicatorplates,whichemployblue/whitescreening.
Figure3.ReporterconstructsinyeaststrainsAH109andY187.StrainAH109isaderivativeofstrainPJ69-2AandincludestheADE2andHIS3markers(Jameset al.,1996).MEL1isanendogenousGAL4-responsivegene.ThelacZreportergenewasintroducedintoPJ69-2AtocreateAH109(A.Holtz,unpublished).TheHIS3, ADE2,andMEL1/lacZ reportergenesareunderthecontrolofthreecompletelyheterologousGAL4-responsiveUASandpromoterelements—GAL1,GAL2,andMEL1,respectively.StrainY187containsthelacZreportergeneundercontroloftheGAL1UAS.
GAL UAS minimal promoter Reporter gene
transcription
Bait protein
DNA-BD
Libraryprotein
AD
GAL1 UAS GAL1 TATA lacZ
Y1�7 Constructs
AH109 Constructs
GAL1 UAS GAL1 TATA HIS3
GAL2 UAS GAL2 TATA ADE2
MEL1 UAS MEL1 TATA lacZ
MEL1 UAS MEL1 TATA MEL1
Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual
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I. Introductioncontinued
Optimizedvectorsfacilitatedownstreamconfirmation
TheMatchmakerSystem3DNA-BDandADfusionvectors,pGBKT7andpGADT7,weredesignedforhigh-levelproteinexpressionandtofacilitateconfirmationofproteininteractions.BaitandlibraryinsertsareexpressedasGAL4fusionswithc-Mycandhemagglutinin(HA)epitopetags,respectively.Theepitopetagseliminatetheneedtogeneratespecificantibodiestoeachnewproteinandallowconvenientidentificationofthefusionproteins.
System3vectorsalsoincludeT7promotersdownstreamoftheGAL4codingsequences.Thesepromotersallowyoutotranscribeandtranslatetheepitope-taggedfusionproteinsin vitro.TheMatchmakerCo-IPKit(Cat.No.630449)allowsyoutoconfirmproteininteractionsthroughanin vitrocoimmunoprecipitation.TheT7promoterisalsoaprimingsiteforDNAsequencing.
Finally,pGBKT7andpGADT7weredesignedtoexpressdifferentbacterialselectionmarkers—kanamycinandampicillin,respectively—tosimplifytheirindependentisolationinE. coli.PurifyingtheplasmidsfromE. coliallowstheisolationofADvectorsafteralibraryscreening.Eachvectoralsocontainsthehigh-copypUCoriginofreplicationtoensurehighyieldsfromplasmidpreparations.These features provide the high quality and quantity of DNA necessary for sequencing andcharacterizinginsertsbyPCRorrestrictiondigests.
Increasedtransformationefficiency
AnotherbenefitofSystem3isthatyeaststrainscarryingpGBKT7aretransformedmoreefficientlythanstrainscontainingotherDNA-BDvectors(Louretet al.,1997;Clontechniques,January1999).ThishighertransformationefficiencyfacilitatestheintroductionofADfusionlibrariesintoyeast,whichmaintainsthecomplexityofthelibraryandincreasestheprobabilityofdetectingnoveltwo-hybridproteininteractions.
Considerations
TheMatchmakerSystemshavebeenusedtoidentifymanydifferenttypesofproteininteractions,includingthosefromprokaryotes,yeast,plants,Drosophila,andmammals.However,allyeasttwo-hybridsystemshavepotentiallimitations:
• SomebaitproteinsmayhaveintrinsicDNA-bindingand/ortranscriptionalactivatingproperties;hence,deletionofcertainportionsofbaitproteinsmayberequiredtoeliminateunwantedactivitybeforetheproteinscanbeusedinatwo-hybridscreen(Bartelet al.,1993b).
• Somehybridproteinsmaynotbestablyexpressedinyeastorlocalizedtotheyeastnucleus.Forproteininteractionsthatnormallyoccuronthecellsurface,aphagedisplaysystemmaybeamoreappropriatechoice.However,thetwo-hybridsystemhasbeenusedtoidentifyextracellularproteininteractions(Ozenberger&Young,1995;Kuoet al.,1992).
• Insomecases,theDNA-BDorADfusionmoietymayoccludethenormalsiteofinteraction,ormayimpairtheproperfoldingofthehybridprotein,andthusinterferewiththeabilityofthetwohybridproteinstointeract(vanAelstet al.,1993).
• Theconditionsinyeastcellsmaynotallowtheproperfoldingorposttranslationalmodifications(suchasglycosylation)requiredforinteractionofsomemammalianproteins.Conversely,thedetectionofaspecificinteractionbetweenmammalianproteinsinayeastsystemdoesnotnecessarily indicatethatthereisacorrespondinginteractionintheproteins’nativeenvironment(Fields&Sternglanz,1994).
• SomeproteininteractionsmaynotbedetectableinaGAL4-basedtwo-hybridsystem,butmaybedetectableusingaLexA-basedsystemsuchastheMatchmakerLexATwo-HybridSystem(Cat.No.K1609-1;Gyuriset al.,1993;reviewedinGolemiset al.,1996;andMendelsohn&Brent,1994),andviceversa.Wecannotpredictwhichsystemwillgivethebestresultsforparticularproteininteractions.
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I. Introductioncontinued
table i. list of abbreviations
Two-HybridTerminology
AD/library AfusionoftheGAL4ADwithalibrarycDNA/protein.
DNA-BD/bait AfusionoftheGAL4DNA-BDwithabaitcDNA/protein.
YeastPhenotypes
Ade–,His–,Leu–,orTrp– Requiresadenine(Ade),histidine(His),leucine(Leu),ortryptophan (Trp)inthemediumtogrow;isauxotrophicforatleastoneofthese specificnutrients.
Ade+ ExpressestheADE2 reportergene;i.e.,doesnotrequireAdeinthe mediumtogrow.
His+ ExpressestheHIS3 reportergene;i.e.,doesnotrequireHisinthe mediumtogrow.
LacZ+ ExpressesthelacZreportergene;i.e.,ispositiveforβ-galactosidase activity.
Mel1+ ExpressestheMEL1reportergene;i.e.,ispositiveforα-galactosi daseactivity.
Miscellaneous
Ade2p Proteinencodedbytheyeast ADE2gene.
3-AT 3-amino-1,2,4-triazole;acompetitiveinhibitoroftheHis3protein.
CHX Cycloheximide
DO Dropout(supplementorsolution);amixtureofspecificaminoacids andnucleosidesusedtosupplementSDbasetomakeSDmedium; DOsolutionsaremissingoneormoreofthenutrientsrequiredby untransformedyeasttogrowonSDmedium.
His3p ProteinencodedbytheyeastHIS3 gene.
SDmedium MinimalSyntheticDropoutmedium;comprisedofanitrogenbase, acarbonsource(glucoseorgalactose),andaDOsupplement.
YPH YeastProtocolsHandbook(PT3024-1)
CompatibleMatchmaker™products
• Pretransformed Matchmaker™ cDNA Libraries provide a high-quality library previouslytransformedintoY187.SimplymatetoAH109thathasbeentransformedwithyourbait.
• Matchmaker™GAL4cDNALibrariesprovideaconvenientmeanstoquicklyscreenahigh-qualitypremadecDNAlibrary.
• pBridgeTMThree-HybridVector(Cat.No.630404)allowsyoutoinvestigateternaryproteincomplexes(Tirode et al.,1997).pBridgeallowsexpressionoftheDNA-BD/baitandathirdprotein.Thethirdproteinisonlyexpressedintheabsenceofmethionine.
• TheMatchmaker™Co-IPKit(Cat.No.630449)providesreagentsforquicklyandindependentlyconfirmingproteininteractionsthroughanin vitrocoimmunoprecipitation.
• Matchmaker™Antibodiesallowyoutoeasilydetectfusionproteins.SeeRelatedProductsfordetails.
•ThepCMV-Myc&pCMV-HAVectorSet(Cat.No.631604)allowsin vivocoimmunoprecipitationsinmammaliancells.
• TheMammalianMatchmaker™Two-HybridAssayKit(Cat.No.630301)isidealforconfirmingproteininteractionsinmammaliancells.
• X-α-Gal(Cat.No.630407)allowsyoutodetect α-galactosidaseactivity.
Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3247-1 � Version No. PR742219
II.Overview:AYeastTwo-HybridScreen
Figure4.Overviewofperformingayeasttwo-hybridscreen.TheappropriateUserManualsectionsareindicated.
Construct DNA/bait
Section VIII.A
Select Transformants
High Stringency: SD/–Ade/–His/–Leu/–Trp/X-α-GalMedium Stringency: SD/–His/–Leu/–Trp
Low Stringency: SD/–Leu/–TrpSection IX.C
(1 week) (1–3 weeks)
(5 days–2 week)
(3 days–1 week)
(3 hr)
(1 week–3 months)
Construct AD/target
Section VIII.A
Construct or Obtain AD/Library
Section VIII.B
Titer and Amplify Library
Appendix B & C
Test for Autonomous Activation
Section VIII.C
Transform AH109
Use sequential or simultaneous methodsSection IX.B
Verify Protein Interactions
Section X
(3 days–1 week)
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III. ListsofComponents
A. Matchmaker™Two-HybridSystem3 Storeallyeaststrainsat–70°C.
StoresequencingprimersandplasmidDNAat–20°C.
• 50 µl pGBKT7CloningVector(0.1µg/µl)
• 50 µl pGADT7CloningVector(0.1µg/µl)
• 50 µl pGBKT7-53ControlVector(0.1µg/µl)
• 50 µl pGBKT7-LamControlVector(0.1µg/µl)
• 50 µl pGADT7-TControlVector(0.1µg/µl)
• 50 µl pCL1ControlVector(0.1µg/µl)
•0.5 ml AH109Saccharomyces cerevisiaeinYPDAmedium/25%glycerol.
•0.5 ml Y187Saccharomyces cerevisiaeinYPDmedium/25%glycerol.
• 40 µl T7SequencingPrimer(0.1µg/µl) 5'–TAATACGACTCACTATAGGGC–3'(21-mer)
• 40 µl 3'DNA-BDSequencingPrimer(0.1µg/µl) 5'–ATCATAAATCATAAGAAATTCGCC–3'(24-mer) • 40 µl 3'ADSequencingPrimer(0.1µg/µl) 5'–AGATGGTGCACGATGCACAG–3'(20-mer) • 10g –Leu/–TrpDropoutSupplement • 10 g –Ade/–His/–Leu/–TrpDropoutSupplement • 1 ml HerringTestesCarrierDNA,denatured • VectorInformationPackets(PT3248-5&PT3249-5)
B. Matchmaker™GAL4cDNA&GenomicLibraries Storeallcomponentsat–70°C.
Dividethelibraryculturesinto100µlaliquots,andstoreat–70°C.Avoidmultiplefreeze/thawcycles.
Retiterandamplifythelibrarybeforeuse(AppendicesBandC).
Theappropriatevectorinformationisprovidedintheaccompanyingvectorinformationpacket.
•2x1.0ml PlasmidLibraryCultureinLBbroth/25%glycerol
• 0.5 ml AH109Saccharomyces cerevisiaeinYPDAmedium/25%glycerol.
• 0.5 ml CG-1945Saccharomyces cerevisiaeinYPDmedium/25%glycerol.
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IV.AdditionalMaterialsRequired
Thefollowingreagentsandmaterialsarenotsuppliedbutarerequired.Recipesforthesemateri-alsareprovidedinAppendixAandtheYPH.
•YPDAortheappropriateSDliquidmedium
•Sterile1XTE/LiAc(Prepareimmediatelypriortousefrom10Xstocks)
•TEbufferorsterile,distilledH2O
•Appropriatesteriletubesorflasksfortransformations.
•AppropriateSDagarplates Notes: • Preparetheselectionmediaandpourtherequirednumberofagarplatesinadvance. • AllowSDagarplatestodryatroomtemperaturefor2–3daysorat30°Cfor3hrpriortoplatinganytrans-
formationmixtures.Moisturedropletsontheagarsurfacecancauseunevenspreading.
• Yeastmaker™YeastTransformationSystem(Cat.No.630439)providesallthenecessaryreagentsforyeasttransformations.Note:
BoilthecarrierDNAfor20minandquicklycoolitonicejustpriortouse.
• SterilePEG/LiAcsolution(Prepareimmediatelypriortousefrom10Xstocks)
• 100%DMSO(Dimethylsulfoxide;SigmaCat.No.D-8779)
• 1XTEbuffer
• Sterile glass rod, bent pasteur pipette, or 5-mm glass beads for spreading cells onplates.
•65%Glycerol/MgSO4solutionforthelow-stringency procedure.
•X-α-Gal(Cat.No.630407)
•
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V.YeastStrains&Phenotypes
ThissectionprovidesdetailedphenotypesoftheyeaststrainsincludedwithMatchmakerSystem3andMatchmakerGAL4cDNAandGenomicLibraries.Foradditionalinformationonthegrowthandmaintenanceofyeast,seetheYPH,ChapterIII.WealsorecommendGuthrie&Fink’sGuide to Yeast Genetics and Molecular Biology (1991)andHeslot&Gailardin’sMolecular Biology and Genetic Engineering of Yeasts (1992).
A. YeastHostStrains ThecompletegenotypesofAH109,Y187,andCG-1945areprovidedinTableII.Allstrainsare
gal4–andgal80–;thispreventsinterferenceofnativeregulatoryproteinswiththeregulatoryelementsinthetwo-hybridsystem.
1.UseAH109asthehoststrainifyouplantoscreenanAD/libraryusingHIS3,ADE2,andMEL1.
2.System3UsersOnly:UseY187asthehoststrainifyouplantotestforaninteractionbetweentwoknownproteinsusingthe lacZreporteronly. Inaddition,useY187asamatingpartnertoverifyproteininteractions.
3.LibraryUsersOnly:UseCG-1945asthehoststrainifyouplantoseparateDNA/baitandAD/library plasmids by cycloheximide counterselection. Alternatively, use pGBKT7toconstructyourbait.ThisDNA-BDvectorcontainsakanamycin resistancemarker;therefore,vectorscanbeseparatedinE. coli withoutcycloheximidecounterselection.
table ii. matchmaker™ yeast strain genotypes Strain Genotype References
AH109 MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, Jameset al.,1996; gal4Δ,gal80Δ, LYS2 : : GAL1UAS-GAL1TATA-HIS3, A.Holtz,unpublished GAL2UAS-GAL2TATA-ADE2, URA3 : : MEL1UAS-MEL1 TATA-lacZ
Y187 MATα,ura3-52,his3-200, ade2-101, trp1-901, Harperet al.,1993 leu2-3,112,gal4Δ,met–,gal80Δ, URA3 : : GAL1UAS-GAL1TATA-lacZ
CG-1945 MATa,ura3-52,his3-200, ade2-101,lys2-801, Feilotteret al.,1994; trp1-901, leu2-3,112,gal4-542,gal80-538,cyhr2, C.Giroux,pers.comm. LYS2 : : GAL1UAS-GAL1TATA-HIS3,
URA3 : : GAL4 17-mers(x3)-CYC1TATA-lacZ B. Phenotypes 1.NutritionalRequirements Toverifyphenotypesandtobecomefamiliarwiththeyeaststrains,testthemforthe
phenotypesshowninTableIII.
a. Streakeachstrainontoadenine-supplementedYPD(YPDA)plates.Incubateat30°Cfor3–5days.Propagateadditionalculturesonlyfromisolatedcolonies.
Note:Thestockmayberefrozenseveraltimeswithoutsignificantlydecreasingviability.
b. Usingasterilelooportoothpick,streak3–4coloniesontotheindicatedSDselectionplates.
c. Incubateplatesat30°Cfor4–6days;yeaststrainsgrowmoreslowlyonSDselectionmediathanonYPDA.
d. SealstockplatewithParafilm,andstoreat4°C.
table iii. matchmaker™ yeast strain phenotypes Strain SD/–Ade SD/–Met SD/–Trp SD/–Leu SD/–His SD/–Ura YPDAYPD/CHX
AH109 – + – – – + + –
Y187 – – – – – + + –CG-1945 – + – – – + + +
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V.YeastStrains&Phenotypescontinued
2.ColonyColorandSize a. Y187andCG-1945carrythe ade2-101mutation.Onmediumwithlowamountsof
adenine,thecolonieswillturnpinkafterafewdaysandmayturndarkerasthecolonyages.Thesecoloniesgrowto>2mmindiameter.However,small(<1mm)whitecolonieswillformatarateof1–2%duetospontaneousmutationsthateliminatemitochondrialfunction(Holm,1993).Avoidthesewhitecolonieswheninoculatingcultures.
IntheabsenceofGAL4,AH109alsoexhibitstheade2-101 phenotype.However,inthepresenceofproteininteractions,theADE2 markercomplementsincistheAH109ade2-101phenotype.
b. WhentransformedwithpAS2-1,oranypAS2-derivedplasmid,CG-1945growsmoreslowlyandformsnoticeablysmallercoloniesthanuntransformedCG-1945.
3.AntibioticResistance CG-1945iscycloheximideresistant.WhenmakingcompetentCG-1945cells,useliquid
YPDmediumwithoutcycloheximide.
4.MEL1 and lacZ ReporterGeneExpressionLevels a. InresponsetoGAL4activation,AH109andY187secreteα-galactosidase,whichcan
bedetectedonmediumcontainingX-α-Gal(Ahoet al.,1997). b. InresponsetoGAL4activation,Y187exhibitsahigherlevelofinducedβ-galactosidase
activitythanbothAH109andCG-1945.ThisisbecauseofdifferencesinthestrengthsofthelacZpromoterconstructs.InY187,lacZisundercontroloftheintactGAL1UAS;inAH109andCG-1945,lacZisundercontroloftheweaker MEL1UASandUASG17-merconsensussequence,respectively.Therefore,useliquidculturesofY187forquantitativeβ-galactosidaseassays.For further informationonβ-galactosidaseassays,see theYPH.
5.LeakyHIS3 Expression a. 3-AT isacompetitiveinhibitoroftheyeastHIS3 protein(His3p).3-ATisusedto
inhibitlowlevelsofHis3pexpression,andthus,tosuppressbackgroundgrowthonSDmediumlackingHis(Fields,1993;Durfeeet al.,1993).
b. CG-1945transformantsaresuppressedbytheadditionof5–15mM3-AT. Ingeneral,AH109doesnotrequire3-AT.However,ifyourDNA/baitproducesbackground
growthonSD/–His/–Trpplates,youwillneedtooptimizetheconcentrationof3-AT. Tooptimizethe3-ATconcentration,platecellstransformedwithyourDNA-BD/bait
plasmidonSD/–His/–Trpplatescontaining0,2.5,5,7.5,10,12.5,and15mM3-AT.Usethelowestconcentrationof3-ATwhich,afteroneweek,allowsonlysmall(<1mm)coloniestogrow.
c. Ahighconcentrationof3-ATinthemediumcankillfreshlytransformedcells.Thus,ifyouwishtouseexcess3-ATtoselectonlyverystrongtwo-hybridinteractions,werecommendusingthelow-stringencyselectionprotocol.
6.Clumping Forunknownreasons,strainCG-1945oftenclumpsinliquidculture.Disperseclumps
byvortexingvigorously.
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VI.ControlVectors
MatchmakerTwo-HybridSystem3providespositiveandnegativecontrolvectors.VectorinformationisprovidedinTableIV.
A. PositiveControls pCL1 encodes the full-length, wild-type GAL4 protein and provides a positive control for
α-galactosidaseand β-galactosidaseassays.
pGBKT7-53andpGADT7-TencodefusionsbetweentheGAL4DNA-BDandADandmurinep53andSV40largeT-antigen,respectively.p53andlargeT-antigeninteractinayeasttwo-hybridassay(Li&Fields,1993;Iwabuchiet al.,1993).
B. NegativeControl pGBKT7-LamencodesafusionoftheDNA-BDwithhumanlaminCandprovidesacontrolfor
afortuitousinteractionbetweenanunrelatedproteinandeitherthepGADT7-TcontroloryourAD/libraryplasmid.LaminCneitherformscomplexesnorinteractswithmostotherproteins(Bartelet al.,1993b;S.Fields,pers.comm.;Ye&Worman,1995).
table iv. matchmaker™ two-hybrid system 3 vectors
Fusion Epitopea Yeastselection Bacterialselection
CloningvectorspGBKT7 DNA/bait c-Myc TRP1 kanamycinpGADT7 AD/library HA LEU2 ampicillin
ControlvectorspCL1 GAL4 LEU2 ampicillinpGADT7-T AD/T-antigen HA LEU2 ampicillinpGBKT7-53 DNA-BD/p53 c-Myc TRP1 kanamycin
pGBKT7-Lam DNA-BD/laminC c-Myc TRP1 kanamycinaHA=hemagglutinin
Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3247-1 14 Version No. PR742219
YoucanusepremadeMatchmakerGAL4cDNAandGenomicLibrarieswithallMatchmakerGAL4-basedsystems.
A. LibraryConstruction cDNAlibrariesarepreparedusingamodifiedGubler&Hoffmanprocedure(1983).
cDNAPrimingMethods • Oligo(dT)primingeliminatesthesynthesisoflengthypoly(dT)regionsandensuresthat
full-lengthclonesand3'endswillbewell-representedinthelibrary(Chenchiket al.,1994;Borson,et al.,1992;Moqadam&Siebert,1994).
• Oligo(dT)+random-primingmayleadtoagreaterrepresentationofallportionsofthegene, includingamino-terminaland internaldomains,regardlessofmRNAsecondarystructure;randomprimingalsogeneratesawidersize-rangeofcDNA.
• Unidirectionallibrariesaremadewitholigo(dT)primersthathaveonevector-compatiblerestrictionenzymesite.Theothersiteisadded(withstickyends)bytheadaptorthatisligated to thecDNA.Thus,digestionwithone restrictionenzymeensures thecDNA'sproperorientationwhenligatedtoavectorthathasbeendigestedwiththeappropriatetwoenzymes.
AdaptorsandLinkers PleaserefertotheProductAnalysisCertificate(PAC)forinformationonthespecificadaptor
orlinkerusedintheconstructionofyourMatchmakerLibrary.
Notes:
• TheopenreadingframeoftheinsertstartsatthecodonimmediatelyfollowingtheC-terminalcodon(a.a.881)oftheGAL4AD,notwithintheadaptor.
• IfanEcoRIlinkerisused,thecDNAismethylatedtoprotectanyinternalEcoRIsites.
• Ifanadaptorisusedintheconstructionofnondirectionallyclonedlibraries,nomethylationorrestrictionenzymedigestionofthecDNAisrequired;therefore,anyinternalEcoRIsitespresentinthecDNAwillnotbecut.
• Ifanadaptorisusedintheconstructionofunidirectionallyclonedlibraries,thecDNAismethylatedtoprotectthealternativesite.
• IfthelibraryissynthesizedusingEcoRI/NotI/SalIadaptors,youmayexcisetheinsertsfromthevectorusingsiteswithintheadaptor.
cDNASizeFractionation Theadaptor-ligateddouble-strandedcDNAissize-fractionatedtoremoveunincorporated
primers,unligatedadaptors,andadaptordimers;thisprocessalsoremoveslow-molecularweight(<400bp)incompletecDNAs.
InsertSizeRangeandAverageInsertSize SizesaredeterminedbyrunningthecDNAonagelpriortocloning,andcomparingthe
profiletoMWsizemarkers.
LibraryAmplification UnlessotherwisestatedonthePAC,alllibrariesareamplifiedonce.
VII. Matchmaker™GAL4cDNA&GenomicLibraries
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Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual
B. LibraryQualityControlInformation ThefollowinginformationisprovidedonthePAC.Thesedatawereobtainedatthetimeof
libraryconstruction.
NumberofIndependentClones Thenumberof independentclones isestimatedbeforeamplification.Most librariesare
guaranteedtohaveatleast1x106independentclones.
LibraryTiter Librarytiterisdeterminedafteramplificationandmustbe>108cfu/mlforplasmidlibraries.
InsertSizeAnalysis Theinsertsizeof15randomlyselectedclonesisdeterminedbyPCRamplificationusing
insertscreeningprimers.
SequenceRepresentation Sequencerepresentationisevaluatedbycolonyhybridizationusingagene-specificprobe.
AllHumanMatchmakercDNALibrariesmustshowaminimum β-actinfrequencyof0.1%,andallothermammalianMatchmakercDNALibrariesmustshowaminimum β-actinfrequencyof0.05%.NonmammaliancDNAlibrariesarescreenedwithaubiquitouslyexpressedspecies-specificprobe.
Note:Thefrequencyofβ-actinpositiveclonesvariesamonglibrariesmadewithRNAfromdifferenttissuesandspecies.Afrequencyof>0.1%inahumancDNAlibrarysuggestsareasonablyhighprobabilityoffindingararetranscript(Hagenet al., 1988).FornonhumanmammaliancDNAlibraries,afrequencyof0.05%suggestsareasonablyhighprobabilityoffindingararemessage(Clontechobservations,unpublished).
PresenceofGenomicDNAorrRNASequencesincDNALibraries ThepurifiedpolyA+RNAusedtoconstructMatchmakercDNAlibrariesisnottreatedwith
DNase,duetopotentialdegradationbycontaminatingRNaseactivity.Therefore,thepolyA+preparationmayhave<1%ofgenomicDNAand<5%ofrRNA.
PCR-basedSequenceScreening • HumancDNAlibraries Arepresentativesample(upto107cfuor>107pfu)ofthetotallibraryisusedasatemplate
inaPCRreactionwithhumanβ-actinPCRprimers.Theseprimersamplifya1.1-kbfragmentlocatedatthe5'endofthegene.AsampleofthelibrarymayalsobeusedasatemplateforPCRamplificationofG3PDHandtransferrinreceptorcDNAfragments.
• NonhumanmammaliancDNAlibraries Arepresentativesample(upto107cfuor>107pfu)ofthetotallibraryisusedasatemplate
inaPCRreactionwithβ-actinPCRprimers.Species-specificPCRprimersareusedforMouseandRatMatchmakercDNALibraries;humanprimersareusedforothernonhumanmammalian libraries. Mouse and rat libraries may also be used as templates in PCRreactionsusingspecies-specificG3PDHprimers.
• NonmammaliancDNAlibraries Arepresentativesampleofthetotallibrary(containingupto107cfuor>107pfu)isused
as a template in a PCR reaction using the appropriate species-specific primers for aubiquitouslyexpressedgene.
VII.Matchmaker™GAL4cDNA&GenomicLibrariescontinued
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A. ConstructFusionGenes Thefollowingisabriefprotocolonconstructinggenefusions.Formoredetailedinformation,
see Sambrook et al. (1989).The orientation and reading frame of each fusion must bemaintainedinordertoexpressfusionproteins.
• Youcangenerateafusiongeneifcompatiblerestrictionsitesarepresentinthetestgenesandthecorrespondingvector. Ifnot,generatethegenefragmentbyPCRwithusefulrestrictionsitesincorporatedintotheprimers(Scharf,1990).ArestrictionsiteattheendofagenecanoftenbechangedintoadifferentsiteorputintoadifferentreadingframeusingaPCRprimerthatincorporatesthedesiredmutation.
• Ifyouareinvestigatingtwoknowngenes,useeithervector—unlessonehasanactivationorDNA-bindingactivitythatwouldinterferewiththeproperfunctioningofthetwo-hybridsystem.
1.Purifythegenefragment,whethergeneratedbyPCRorcutoutofaplasmid. Note:WerecommendtheNucleoSpinExtractionKit(Cat.No.635961)forrapidisolationofDNAfragments.
2.DigesttheDNA-BDorADvectorwiththeappropriaterestrictionenzyme(s),treatwithphosphatase,andpurify.
3.Ligatetheappropriatevectorandinsert.TransformligationmixturesintoE. coli. 4.Identifyinsert-containingplasmidsbyrestrictionanalysisorPCRusingtheMatchmaker
InsertScreeningAmplimerSet(Cat.No.630433). 5.UsetheSequencingPrimersincludedwithMatchmakerSystem3tochecktheorientation
andreadingframeofthejunctions.
B. ObtainorConstructanADFusionLibrary PremadeMatchmakercDNALibrariesandPretransformedcDNALibrariesfromavarietyof
tissuesandspeciesareavailablefromClontech.Alternatively,constructanADfusionlibraryinpGADT7usingeitherintronlessgenomicDNAorcDNAsuchthatatleast106differenthybridproteinswillbeexpressed(Ausubelet al.,1995).
Notes: • Two-hybridlibrariesareusuallyconstructedintheADvectorratherthantheDNA-BDvector.Fusingrandomproteins
toaDNA-BDwillproduceamuchlargerpercentageoffusionsthatfunctionasautonomoustranscriptionalactivators(Ma&Ptashne,1987).
• OtherGAL4ADvectorsarecompatiblewithMatchmakerTwo-HybridSystem3,providedtheycarrytheLEU2 nutritionalmarker.
1.Amplificationofpremadelibraries ObtainpremadelibrariesasE. coli transformants,notaspurifiedDNA.Amplifythelibraryto
produceenoughplasmidDNAtoscreenthelibraryinyeast.IfyouhaveobtainedaMatchmakercDNALibrary, followtheamplificationprotocolprovided inAppendixC. Ifyouhaveobtainedalibraryfromanothercommercialsource,followthemanufacturer’sinstructions.
2.ConstructionofcDNAlibraries UseanystandardmethodforgeneratingcDNA(Sambrooket al.,1989;Ausubelet al.,
1995).Fordetailedinformationregardingconstructionoftwo-hybridcDNAlibraries,seeVojteket al.(1993),Durfeeet al.(1993),Dalton&Triesman(1992),andLubanet al.(1992).
Besuretoreservea1.0-mlaliquotofyourlibrary,frozenin25%glycerol,sothatyoucangobackandamplifyitlater.
YoucanalsoconstructgenomicDNAlibrariesfromanorganismwhosegenomecontainsfeworno introns,suchasbacteriaoryeast.pGADT7containsauniqueBamHIsiteforconstructingaSau3AI-digestedgenomicDNAlibraryandauniqueCla Isite forconstructingalibraryaccordingtoJameset al.(1996).Note:AprocedurefortiteringaplasmidlibraryinE. coliisinAppendixB.
VIII.PreparingforaYeastTwo-HybridScreen
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C. VerifythatConstructsDoNotActivateReporterGenes 1.IndependentlytransformyourDNA-BDandADfusionconstructsintostrainAH109.Assay
thetransformantsforMEL1 activationby selectingfortransformantsonSD/–Trp/X-α-GalandSD/–Leu/X-α-Gal,respectively.Performpositiveandnegativecontrolsinparallel(SeeSectionIX,TableVI).
Ifapartner isknownforyourDNA-BD/bait,use it tocheckwhetheran interaction isdetectablebeforeinvestinginalibrarysearch.
2.Ifthetransformantcoloniesarewhite,preparestockplatesandliquidculturesforfreezing. Iftransformantcoloniesareblue,seeSectionXIforpossiblesolutions.
D. VerifyProteinExpression 1.IndependentlytransformtheDNA-BDandADfusionconstructsintostrainAH109. 2.PrepareWesternblotsfromthetransformantsandprobetheblotswithantibodiesto
thec-MycandHAepitopetags(Cat.Nos.631206,631207)ortheGAL4DNA-BDandADMonoclonalAntibodies(Cat.Nos.630403,630402.Useuntransformedyeastasacontrol.SeetheYPH(SectionIV)forprotocolsonpreparingproteinextractsfromyeast.
Note:Usingpolyclonalantibodiesmayresultinmultiplecross-reactingbands.
Alternatively,usetheT7promotertotranscribeandtranslatetheepitope-taggedfusionproteinsin vitro.ConfirmproteintranscriptionandtranslationbycoimmunoprecipitationusingtheMatchmakerCo-IPKit(Cat.No.630449).
VIII.PreparingforaYeastTwo-HybridScreencontinued
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IX. LibraryTransformation&ScreeningProtocols
Inthissection,weprovidedetailedprotocolsforpolyethyleneglycol/lithiumacetate(PEG/LiAc)-mediatedtransformationofyeast(Itoet al.,1983;Schiestl&Gietz,1989;Hillet al.,1991;Gietzet al., 1992).
The procedures described here are for library screens using strain AH109 only. If you choose to use strain CG-1945, follow the same general procedures and plate on SD/–His/–Leu/–Trp. DO NOT PLATE STRAIN CG-1945 ON MEDIA LACkING ADENINE; IT wILL NOT GROw.
A. TransformationScales Weprovideprotocolsforsmall-,large-,andlibrary-scaletransformations.TableVcompares
thetransformationmethods.
• Usesmall-scaletransformationsto: – VerifythattheDNA-BD/baitdoesnotautonomouslyactivatereportergenes – LookfortoxicityeffectsofDNA-BD/baitonhost – Performcontrolexperiments – TransformtheDNA-BD/baitforsequentialtransformations • Uselarge-scaletransformationswhenyouarelearningtwo-hybridscreening,orwhen
youdonothaveenoughDNAforalibrary-scaletransformation(TableV).Youcanperformeithersequentialorsimultaneoustransformations.
• In a sequential transformation, the DNA-BD/bait plasmid is introduced through asmall-scaletransformation;selectedtransformantsarethengrownupandtransformedwiththeADfusionlibrarythroughalarge-scaletransformation.AsequentialtransformationmaybepreferredbecauseitusessignificantlylessplasmidDNAthanasimultaneouscotransformation(TableV).
• Asimultaneouscotransformationisgenerallypreferredbecauseitiseasiertoperformthanasequentialtransformation—andbecauseoftheriskthatexpressionoftheDNA-BD/baitproteinmaybetoxictothecells.IftheDNA-BD/baitproteinistoxic,clonesarisingfromspontaneousdeletionsintheDNA-BD/baitplasmidwillhaveagrowthadvantageandwillaccumulateattheexpenseofclonescontainingintactplasmids.
• Library-scaletransformationsarepreferredwhenscreeninganAD/library. Inthecaseoflargeandlibraryscalesimultaneouscotransformations,youmustdetermine
thetransformationefficienciesofbothplasmidstogether,aswellasofeachtype ofplasmidindependently.ExamplecalculationsareshowninSectionIX.D.
table v. comparison of two-hybrid transformation methods
Amount No.ofIndep. ofLimiting Amount Transformation Clones Transformation Plasmid ofCells Efficiency Amplified No.ofPlates
Library-scale 100–500µg 8ml 103–104 1x106 50x150-mmLarge-scaleSimultaneous 10–50µg 1.5ml 103–104 1x105 5x150-mmSequential 10–50µg 1.5ml 104–105 1x106 50x150-mmSmall-scale 0.1µg 1.5ml 105 na 1x100-mmna=notapplicableacfuperµgoftheADlibrary.b TotalapproximatenumberoftransformantsexpectedonSD/–Leu/–Trpselectionplatesassumingthat:1)theminimal
amountofplasmidwasused;2)thetransformedcellswereresuspendedinthevolumesrecommendedintheprotocol;3)200µloftransformedcellswerespreadoneachplate;and4)thetransformationefficiencieswereoptimal.
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IX. LibraryTransformation&ScreeningProtocolscontinued
B. YeastTransformationProtocols Tipsforasuccessfultransformation •Usea1–3week-oldcolony(2–3mm)toinoculateeachliquidculture.Ifcoloniesonthe
stockplateare<2mm,useseveralcolonies. Note:Toaidinresuspendingthecells,placethecolonyina1.5-mltubecontaining0.5mlofmediumandvortex
vigorously.Thentransferthecellsuspensiontothecompletevolumeofculturemedium.
• Iftheovernightor3-hrculturesarevisiblyclumped,dispersetheclumpsbyvortexing. • Whenyouarecollectingcellsbycentrifugation,aswingingbucketrotorresultsinbetter
recoveryofthecellpellet. • Titertheoptimalconcentrationof3-ATneededtoeliminatebackgroundgrowthon–His
selectionplates. • For thehighest transformationefficiency,usecompetentcellswithin1hrpreparing
them.Ifnecessary,youcanstorecompetentcellsafterStep11atroomtemperatureforseveralhourswithonlyaminorreductionincompetency.
• Whenperformingsimultaneouscotransformations,thebaitplasmidmustbeusedinexcess,andtheADplasmidorlibrarymustbelimiting.
• Toobtainanevengrowthofcolonies,spreadthetransformationmixtureovertheagarsurfaceuntilallliquidhasbeenabsorbed.Alternatively,use5-mmsterileglassbeads(5–7beadsper100-mmplate;7–9beadsper150-mmdiameterplate)topromoteevenspreading.Tospreadthecolonies,shaketheplatebackandforth—notroundandround.
Belowareprocedures forpreparingcompetentcellsand transformingyeast.Setup thecontrolandexperimentaltransformationslistedinTableV.
TransformationScale
SMALL LARGE LIBRARY 1. Inoculate1mlofYPDAorSDawith several2–3mmcolonies.
2. Vortexvigorouslytodisperseanyclumps.
3. Transfercellstoaflaskcontainingthis volumeofYPDAorSDa: 50ml 50ml 150ml
4. Incubateat30°Cfor16–18hrwithshaking (250rpm)tostationaryphase(OD600>1.5).
5. Transferovernightculture(enoughtoproducean OD600=0.2–0.3)intothisvolumeofYPDA: 300ml 300ml 1L
6. Incubateat30°Cfor3hrwithshaking (230–270rpm).TheOD600willbe0.5±0.1.
7. Placecellsin50-mltubesandcentrifugeat 1,000xgfor5minatroomtemperature.
8. Discardthesupernatantandresuspendcell pelletsbyvortexinginthisvolumeofsterile TEorH2O: 25–50ml 25–50ml 500ml
9. Poolcellscentrifugeat1,000xgfor5min atroomtemperature.
10. Decantthesupernatant.
11. Resuspendthecellpelletinthisvolumeof freshlyprepared,sterile1XTE/LiAc: 1.5ml 1.5ml 8mlb
12. PreparePEG/LiAcsolution. 10ml 10ml 100mlaUseSD/–Trpwhenperformingthesecondtransformationinasequentialtransformationprotocol.bForlibrarycotransformationsonly:removetwo100-µlaliquotsofcompetentcellstoperformcontroltransformations
withpCL1,andpGBKT7-53+pGADT7-T.
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TransformationScale
SMALL LARGE LIBRARY
13. Intheindicatedtube,addandmixthefollowing: 1.5ml 50ml 500ml •DNA-BD/baita 0.1µg 20–100µg 0.2–1.0mg
•AD/library 0.1µg 10–50µg 0.1–0.5mg
•HerringtestescarrierDNA 0.1mg 2mg 20mg
14. Addthisvolumeofyeastcompetentcells: 0.1 ml 1ml 8ml andmixwellbyvortexing.
15. AddthisvolumeofsterilePEG/LiAcsolution: 0.6ml 6ml 60ml andvortexathighspeedtomix.
16. Incubateat30°Cfor30minwith shaking(200rpm).
17.AddthisvolumeofDMSO: 70µl 700µl 7.0ml Mixwellbygentleinversionorswirling. Donotvortex.
18. Heatshockfor15minina42°Cwaterbath. Forlarge-andlibrary-scale,swirloccasionally tomix.
19. Chillcellsonicefor1–2min.
20. Centrifugecellsfor: 5sec 5min 5min
atroomtemperatureat: 14Krpm 1,000xg 1,000xg
21. Removethesupernatant.
22. Resuspendcellsinthisvolumeof1XTEb: 0.5ml 1.0mlor10mlc 10ml
23. ProceedtoSectionIX.Cforplating.
a Forsimultaneouscotransformation,werecommendamolarratioof2:1(DNA-BDvector:ADvector)foroptimaleffi-ciency.Forsequentialtransformations,addeithertheDNA-BDvectorconstructortheADvectorconstruct(notboth).
bIfusingthehighstringencyselectionmethod,resuspendcellsinYPDA.Themediawillaidtheyeastinrecoveryfromtheshockoftransformation,butwillnotadverselyaffectscreening.
cUse1.0mlforsimultaneouscotransformation.Use10mlforthesecondtransformationinasequentialtransformationprotocol.
IX. LibraryTransformation&ScreeningProtocolscontinued
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C. PlatingandScreeningTransformationMixtures YoucanselectAH109transformantsusinghigh-,medium-,orlow-stringencymedia(Figure
5).Lessstringentscreensincreasethenumberoffalsepositives,whilemorestringentscreensmayresultinfalsenegatives.
• High-stringency:PlatetransformationsonSD/–Ade/–His/–Leu/–Trp/X-α-GalmediumtoscreenforADE2, HIS3, and MEL1 expression.Thisscreenvirtuallyeliminatesfalsepositiveinteractions;however,low-affinityproteininteractionsmaybemissed.
• Medium-stringency:PlatelibrarytransformationsonSD/–His/–Leu/–TrpmediumtoscreenforexpressionofHIS3.Plantoscreenatleast1.5–3timesthenumberofindependentclonesinthelibrary.Subsequently,replicaplateHis+coloniesontoSD/–Ade/–His/–Leu/–Trp/X-α-GalmediumtoscreenforADE2andMEL1expression.
• Low-stringency:Performthisscreenifyouarehavingtroublepickinguppositiveclonesorifyoususpectthatyourbaitproteininteractsveryweaklyortransientlywithotherproteins.
PlateoutlibrarytransformationsonSD/–Leu/–TrpmediumtoselecttheDNA-BDandADvectors.Thisselectionstepprovidesaninitialphaseofgrowththatmaximizesplasmidcopynumber,whichresultsinhigherlevelsoffusionprotein.This,inturn,improvesthechancesofdetectingADfusionproteinsthatinteractweaklyortransientlywiththebait.
Thisscreentypicallyresultsinupto1,000candidatecolonies.Therefore,youmustoptimizethe3-ATconcentrationneededtocontrolbackgroundgrowth.Furthermore,alowstringencyscreenmayresultinapopulationpreferenceforclonesexhibitingstrongeractivationoftheHIS3 reporter,andextrastepsmayberequiredtosorttheclonesintogroupsbeforeyouproceedwithfurtheranalysis.
table vi. set up for a two-hybrid library screen
Vectors Scalea SDMinimalMedium Amountto Phenotype Plate(µl)Mel1/LacZHis/AdeControlpCL1 S –Leu 100 Blue +pGADT7-T S –Leu 100 White –+pGBKT7-53 S –Trp 100 White – S –Leu/–Trp 200 Blue + S –Ade/–His/–Leu/–Trp/X-α-Galb 200 Blue +pGADT7-T S –Leu 100 White –+pGBKT7-Lam S –Trp 100 White – S –Leu/–Trp 200 White – S –Ade/–His/–Leu/–Trp/X-α-Galb 200 NoGrowth –
ExperimentalDNA-BD/bait L/Lib –Leuc 100 White –+ADlibrary L/Lib –Trpc 100 White – L/Lib Low:–Leu/–Trpd,e,f 100 White – L/Lib Medium:–His/–Leu/–Trpb,e 200 White/Blue–/+ L/Lib High:–Ade/–His/–Leu/–Trp/X-α-Galb 200 White/Blue–/+aS=smallscale;L=largescale;Lib=libraryscale.bIfnecessary,seeSectionV.B.5forguidelinesonhowmuch3-ATtoadd.cTotestthetransformationefficiencyofeachplasmid,dilute1µlofthetransformationwith100µlofH2O.Spread1µl
onto100-mmSD/–LeuandSD/–Trpplates.dTotestthecotransformationefficiencyspread100µlofa1:1,000,1:100,and1:10dilutiononto100-mmSD/–Leu/–Trp
plates.e Plateatleast1.5–3timesthenumberofindependentcolonies.f Ina low-stringency libraryscreen,use three–Hisselectionplates:onewith theoptimalconcentrationof3-AT;one
witha10–15-mMhigher3-ATconcentrationtocontrolforbackgroundgrowthdue;andonewitha5-mMlower3-ATconcentrationforimprovedgrowthofweakpositives.
IX. LibraryTransformation&ScreeningProtocolscontinued
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IX. LibraryTransformation&ScreeningProtocolscontinued
Figure5.ScreeninganADfusionlibraryusingstrainAH109.Usethestringencyofyourchoicetoscreenforinteractingproteins.Note:highstringencyselectionsresultinfewercolonies,andreducethenumberoffalsepositives.However,weakinteractionsmaybemissed.
Plate culture on SD/–Leu/–Trp to select all cotransformants
Colony growth and blue color indicates an interaction between the two-hybrid proteins
Scrape colonies and make a glycerol stock
(Nearly confluent growth)
Cotransform AH109
DNA-BD/baitMarker: TRP1
AD/fusion libraryMarker: LEU2
Plate culture on SD/–His/–Leu/–Trp
Plate culture onSD/–Ade/–His/–Leu/–Trp/X-α-gal
Medium-stringency
Replica plate to SD/–Ade/–His/–Leu/–Trp/X-α-gal
or
Low-stringency
Plate culture on SD/–Ade/–His/–Leu/–Trp/X-α-gal
High-stringency
1.PlatetransformationmixturesasindicatedinTableV.Platesmall-scaletransformationson100-mmplatesandlarge-andlibrary-scaletransformationson150-mmplates.Withanewbaitandlibrarycombination,predictingtheoptimalmethodisdifficult.Therefore,plateathirdofthetransformationonlow-,medium-,andhigh-stringencyplates.
2.Incubateplatesupside-downat30°Cuntilcoloniesappear. 3.IfscreeninganAD/library,calculatethetransformationefficiencyandestimatethenum-
berofclonesscreened,asdescribedinSectionIX.D.
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IX. LibraryTransformation&ScreeningProtocolscontinued
4.LOW-STRINGENCYPROTOCOLONLY.Harvestthelibrarytransformantsasfollows: a. Chillplatesat4°Cfor3–4hr. b. Add1–5mlofTEbuffer(pH7.0)toeachplate.Carefullyscrapethecoloniesintothe
liquidusingaheat-bent,sterilePasteurpipette.Combineallliquidsinasterile50-mltube,andvortextoresuspendthecells.
Note:Ifthecombinedvolumeistoolarge,reduceitasfollows:centrifugefor5minat1,000xg,removeallbut25–50mlofthesupernatant,andvortextoresuspendthecells.
c. Createaglycerolstockbyaddinganequalvolumeofsterile65%glycerol/MgSO4solution. d. Divideinto1-mlaliquots,andstoreat4°Cforaweekorat–80°Cupto1yr. e. TitertheglycerolstockonSD/–Leu/–Trp(AppendixB).Incubateplatesat30°Cfor
3–4daysoruntilcoloniesareeasytocount.Calculatethecfu/µloflibrary. f. Platetheamplifiedyeastcotransformantsathighdensityoneither: • High-stringencyplates:SD/–Ade/–His/–Leu/–Trp/X-α-gal • Medium-stringencyplates:SD/–His/–Leu/–Trp To compensate for possible errors in the amplified library titer, plate 0.5 x 106
cfuonsomeplatesand2x106cfuonothers.Also,plateappropriatecontrolsforcomparison.
Note: If you plate on medium-stringency plates, you must replica plate to high-stringency plates toeliminatefalsepositives.
g. Incubateplatesupside-downat30°Cuntilcoloniesappear. 5.ChooseAde+/His+/Mel1+coloniesforfurtheranalysis.
Note:After2–3days,someAde+/His+colonieswillbevisibleonthehigh-stringencyplates;however,incubateplatesfor5–10daystoallowweakpositivestogrow.Ignoresmall,palecoloniesthatappearafter2daysbutnevergrowto>2mmindiameter.TrueHis+coloniesarerobustandcangrowto>2mm.Ade+colonieswillremainwhitetopalepink;Ade–colonieswillgraduallyturnreddish-brownandstopgrowing.StrongerADE2expressionwillbewhite,whileweakerexpressionwillbeprogressivelymorered.
6.[Option]Performaβ-galactosidasefilterassay(YPH).
D. Calculations 1.CotransformationEfficiency. Countcolonies(cfu)growingontheSD/–Leu/–Trpdilutionplatethathasbetween30–300cfu:
cfuxtotalsuspensionvol.(µl) =cfu/µgDNA Vol.plated(µl)xdilutionfactorxµgDNAused* *Inacotransformation,thisistheamountoflimiting plasmid,notthetotalamountofDNA.
2.NumberofClonesScreened. cfu/µgxµgoflibraryplasmidused=No.ofclonesscreened Examplecalculation: • 100coloniesgrewonthe1:100dilutiontransformationefficiencycontrolplate(dilution
factor=0.01) •resuspensionvolume=10ml •amountoflibraryplasmidused=100µg
100cfu x (10mlx103µl/ml) =1x104cfu/µg 100µlx0.01x100µg
•1x104cfux100μg=1x106clonesscreened.
3.AmountofDNAtoUse.
Ifyouscreened<106clones,repeatthetransformationusingmoreDNA.CalculatetheamountofDNAtouseintherepeattransformationasfollows:
106clones =µgDNAneeded
No.ofclonesscreened/µgDNAused
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X.Analysis&VerificationofPutativePositiveClones
Thissectionprovidesprotocolsforverifyingproteininteractions.Figure6providesadetailedoverview.
A. RetestPhenotypes TheinitiallibrarycotransformantsmaycontainmorethanoneAD/libraryplasmid,which
cancomplicatetheanalysisofputativepositiveclones. 1.RestreakpositivecoloniesonSD/–Leu/–Trp/X-α-Galplates2–3timestoallowlossofsome
oftheAD/libraryplasmidswhilemaintainingselectivepressureonboththeDNA-BDandADvectors.Incubateplatesat30°Cfor4–6days.Amixtureofwhiteandbluecoloniesindicatessegregation.
2.Replicaplateortransferwell-isolatedcoloniestoSD/–Ade/–His/–Leu/–Trp/X-α-Galplatestoverifythattheymaintainthecorrectphenotype.
3.CollecttherestreakedandretestedAde+/His+/Mel1+coloniesonSD/–Ade/–His/–Leu/–Trpmasterplatesinagridfashion.Incubateplatesat30°Cfor4–6days.Aftercolonieshavegrown,sealplateswithParafilm,andstoreat4°Cforupto4weeks.
B. IsolatePlasmidDNAfromYeast TheYeastmakerYeast Plasmid Isolation Kit (Cat. No. 630441) provides the reagents and
aprotocolforisolatingplasmidDNAfromyeast.TheseproceduresprovideplasmidDNAsuitable for PCR and E. coli transformations.A similar protocol is provided in theYPH.Note:theplasmidDNAisolatedfromeachpositiveyeastcolonywillbeamixtureoftheDNA-BD/baitplasmidandatleastonetypeofAD/libraryplasmid.
Alternatively,youmaywishtotrythedirecttransferofplasmidDNAfromyeasttoE. coli byelectroporation(Marcil&Higgins,1992).
Note:Forthismethod,thetransformationefficiencyofcompetentE. colicellsmustbe>109cfu/mg.
C. SortColoniestoEliminateDuplicates 1.AmplifyAD/libraryinsertsbyPCRandcharacterizePCRproductsbydigestingwitha
frequent-cutterrestrictionenzyme,suchasAlu IorHae III.Analyzefragmentsizesbyagarosegelelectrophoresis;also,runasampleoftheuncutamplifiedinserttocheckformultipleAD/libraryplasmids.Prepareanewmasterplatewitharepresentativeclonefromeachgroup.Ifyouaresatisfiedwiththenumberofuniqueclones,proceedtoStep3.
Notes:ToamplifyAD/libraryinserts,werecommendtheMatchmakerADLDInsertScreeningAmplimerSet(Cat.No.630433)andtheAdvantage2PCRKit(Cat.Nos.639206,639207).
2.IfahighpercentageofthecoloniesappeartocontainthesameAD/libraryinsert,expandyourPCRanalysistoanotherbatchof50colonies.
Alternatively,eliminatetheabundantclonesbyperformingyeastcolonyhybridizationoneachmasterplate.Useavector-freeoligonucleotideprobedesignedfromthesequenceofthemostabundantinsert.Transferarepresentativeofeachtypeofinserttoanewmasterplate.
CAUTION:SomepositivecoloniesmaycontainmultipleAD/libraryplasmids—evenifthecolonyhasbeenrestreakedtwice.Therefore,ifapositivecolonyappearstohavemultipleAD/libraryplasmids,donotimmediatelyeliminatethosethatcontaintheabundantinsert.
3.Prepareaglycerolstockofeachuniquetype,andstorealiquotsat–80°C.
D. RescueAD/LibraryPlasmidsviaTransformationofE. coli. 1.LibraryUsers: • ForstrainCG-1945,usecycloheximide(CHX)counterselectiontoobtaincoloniesthathave
losttheDNA-BDandretainedtheAD.RefertotheYPH(ChapterIX)forthisprocedure. • IfyoutransformedstrainAH109anddidnotuseDNA-BDandADvectorswithdifferent
antibiotic markers, transform KC8 E. coli cells and plate on M9 medium lackingleucine.KC8cellshaveadefectin leuBthatcanbecomplementedbyyeast LEU2.
System 3 Users or Libraries User with a pGBKT7/bait: Transform the yeast-purifiedplasmid DNA into E. coli.To select for transformants containing only theAD/libraryplasmid,plateonLBmediumcontainingampicillin.
2.ToverifythatyouhaveobtainedthesameAD/libraryplasmid,amplifyinsertsbyPCR.Thendigest the fragmentwithAlu IorHae III, and runasmall sampleonan3–4%agarose/EtBrgel.ComparethePCRproductgelprofilesfromE. coliandyeast.
Protocol No. PT3247-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR742219 2�
Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual
X.Analysis&VerificationofPutativePositiveClonescontinued
Figure6.Strategiesforanalyzingandverifyingputativepositiveclones.IfthelibraryscreenwasperformedusingstrainCG-1945,seetheYPHSectionIXfordetailsonhowtosegregatetheDNA-BD/baitandAD/libraryplasmids.
E. RetestProteinInteractionsinYeast Youcanretestproteininteractionsinyeastbyeithercotransformationoryeastmating.A
realinteractionwillbehavelikethecontrolsinTableVI. 1.Cotransformation a. Using thesmall scale transformationprocedure, transform theDNA-BD/baitand
AD/libraryplasmidsintoAH109. b. PlateonSD/–Ade/–His/–Leu/–Trp/X-α-gal. c.Incubateplatesat30°Cuntilcoloniesappear.
Confirm Interaction in Yeast
Cotransform DNA-BD/bait and AD/library plasmids into AH109
orPerform yeast matings
In vitro coimmunoprecipitationusing Matchmaker Co-IP Kit
(Cat. No. 630449)
Retest Ade+/His+/Mel1+(LacZ+) phenotype
Eliminate colonies bearing the same AD/library plasmid by
a) PCR or
b) Colony Hybridization
Isolate plasmids from yeast
Transform plasmids into E. coli and purify DNA
Confirm protein interactionsin mammalian cells
Mammalian Two-HybridAssay Kit
(Cat. No. 630301)
In vivo Co-IP usingpCMV-Myc & pCMV-HA
(Cat. No. 631604)
Additional Two-Hybrid Tests- Switch Vectors- Frameshift Mutations- Site-specific mutations/deletions
Sequence cDNA inserts
Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3247-1 2� Version No. PR742219
X.Analysis&VerificationofPutativePositiveClonescontinued
Figure7.Yeastmatingtoverifyproteininteractions.
(1) DNA-BD (2) DNA-BD/bait (3) DNA-BD/laminC
Master plate with candidate clones1. Transform AH109 with: a) AD/library b) AD vector
Plate on SD/–Leu
2. Inoculate 0.5-ml YPD cultures.
4. Plate on SD/–Leu/–Trp
3. Mate to Y187 transformed with:
5. Replica plate or streak onto SD/–Ade/–His/–Leu/–Trp/X-α-Gal
(4) DNA-BD (�) DNA-BD/bait
AH109 [AD vector]
2.YeastMating Yeastmatingisaconvenientmethodofintroducingtwoplasmidsintothesamehost
cells(Finley&Brent,1994;Harperet al.,1993).IfyouhavemanyAde+,His+,Mel1+/LacZ+positiveclonestoanalyze,itwillbemoreconvenienttohandletheclonesinbatchesof10orsoeach.
a. TransformAH109withtheAD/libraryandADplasmid,andselectonSD/–Leu. b. TransformY187(orasuitableMatαstrain)withthefollowingthreeplasmids,and
selectonSD/–Trpplates: i. DNA-BD ii. DNA-BD/bait iii. pGBKT7-Lam c. ForeachcandidateAD/libraryplasmidtobetested,setuptheyeastmatingsindicated
inFigure7usingtheTrp+andLeu+transformantsobtainedinStepsa&babove. d. RefertotheYPH,ChapterIXformatingprocedures.Toselectfordiploids,spread
matingmixturesonSD/–Leu/–Trpplatesasdirected. e. Streakorreplica-platetoSD/–Ade/–His/–Leu/–Trp/X-α-gal.TruepositivesareAD/library
clones exhibiting reporter gene expression only when theAD/library plasmid isintroducedbymatingwiththeplasmidencodingtheDNA-BD/baitprotein.Discardanyβ-galactosidase-positivecoloniescontainingtheAD/libraryplasmidalone.
F. In vitro Analysis
TheMatchmakerCo-IPKit(Cat.No.630449)allowsyoutoconfirmproteininteractionsquicklyandindependentlyviaan in vitrocoimmunoprecipitation.TheCo-IPKitworkswithallGAL4-basedMatchmakerSystemandLibraryvectors.BecauseSystem3vectorsalreadycontainT7promotersandepitopetags,youcanusethemdirectlyinin vitro transcription/translationreactions.ForallotherGAL4-basedvectors,youmustfirstusetheCo-IPPrimerstoamplifyinsertsinordertoincorporatetheT7promotersandepitopetags.TheCo-IPKitalsoprovidesc-MycandHAantibodiesforprecipitatinginteractingproteins.
1.Transcribeandtranslatetheepitope-taggedfusionproteins in vitrousingtheT7promotersintheADandDNA-BDvectors.Note:theT7promoterislocateddownstreamoftheGAL4codingsequence;hence,theGAL4domainsarenottranscribed.
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X.Analysis&VerificationofPutativePositiveClonescontinued
2.Coimmunoprecipitatetheepitope-taggedfusionproteinsusingc-MycandHAantibodies(Durfeeet al.,1993;Zhanget al.,1993).
If the fusion proteins do not coimmunoprecipitate, use other means to confirm theinteraction. Note: protein interactions with weak affinities may escape detection bycoimmunoprecipitation.SeePhizichy&Fields(1995)fordetailsonmoresensitivedetectionmethods.Furthermore,theADfusionproteinsmaypotentiallynotbein-framewiththeepitopetag. See Section X.G. 2–5 for further recommendations.
G. SequenceAD/LibraryInserts UseonlyDNAisolatedfromE. coli. 1.SequenceinsertsinthepositiveAD/libraryplasmidsusingthe3'ADSequencingPrimer
andT7SequencingPrimerprovidedwithSystem3.Verifythepresenceofanopenreadingframe(ORF)fusedtotheGAL4ADsequence,andcomparethesequencetothoseinGenBank,EMBL,orotherdatabases.
2.If yoursequencing results revealapeptide<10-aminoacids fused to theAD—ornofusionpeptideatall—keepsequencingbeyondthestopcodon.YoumayfindanotherORF.NontranslatedgapsupstreamofORFinsertsaremostcommonlyfoundinyeastgenomiclibraries,whereintercistronicregionsareveryshort.SuchgapscanalsooccurincDNAlibraries,duetothecloningofaportionofthe5'untranslatedregionofthemRNAalongwiththecodingregioninthecDNA.Ifthelibrarywasbuiltinahigh-levelexpressionvectorsuchaspGADT7,pGADGH,orpACT2,aWesternblotwillrevealthepresenceandsizeofanADfusionprotein.
3.Duetooccasionaltranslationalread-through,twodifferentORFsmayoccasionallybeexpressedasafusionwiththeAD,eventhoughanontranslatedgapcomesbetweenthem.
4.IfyoursequencingresultsfailtorevealanyORFinframewiththeADcodingregion,thepositivelibraryclonecouldbetranscribedinthereverseorientationfromacrypticpromoter within the ADH1 terminator (Chien et al., 1991). Such proteins apparentlyfunctionastranscriptionalactivatorsaswellasinteractingwiththebaitprotein.
5.Yeastalsoallowtranslationalframeshifts.AlargeORFinthewrongreadingframemayactuallycorrespondtotheexpressedprotein.
H. In vivo Analysis Ifthefusionproteinscoimmunoprecipitate,confirmfunctionalanalysis in vivo througheither
acoimmunoprecipitationoraMammalianTwo-HybridAssay(Cat.No.630301). 1.We recommend the pCMV-Myc & pCMV-HAVector Set (Cat. No. 631604) for in vivo
coimmunoprecipitationinmammaliancells.TheCMVpromoterinthesevectorsallowsconstitutiveexpressionofthebaitandlibrarycDNAinawidevarietyofmammaliancelltypes.AllMatchmakerGAL4-basedvectorsarecompatible;therefore,youcaneasilytransferyourbaitandlibraryinsertsintopCMV-MycandpCMV-HA.
2.TheMammalianTwo-HybridAssayKit is idealforconfirmingproteininteractionsviatwo-hybridinteractionsinmammaliancells.Proteinsaremorelikelytobeintheirnativeconformationsandtohavetheappropriateposttranslationalmodifications;therefore,resultsaremorelikelytorepresentbiologicallysignificantinteractions.
I. AdditionalTwo-HybridTests 1.TransferthelibraryinsertfromtheADtotheDNA-BDvectorandviceversa,andthen
repeatthetwo-hybridassay(Chienet al.,1991;vanAelstet al.,1993).Youshouldstillbeabletodetecttheinteraction.
2.CreateaframeshiftmutationjustupstreamofthelibraryinsertintheADplasmidbycuttingattheMluIsite,fillingintheoverhangs,andthenreligating(Bendixenet al.,1994).Repeatthetwo-hybridassay;youshouldnotbeabletodetecttheinteraction.
3.Generatesite-specificdeletionorsubstitutionmutantsandrepeatthetwo-hybridassay.Assaytherelativestrengthoftheinteractionsusingaquantitativeβ-galactosidaseassay(YPH).
Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3247-1 2� Version No. PR742219
XI.TroubleshootingGuide
DNA-BD/baitactivatesreportergenes
Excessivebackground
Lowtransformationefficiency
Iftwotestproteinsarebeingassayed,switch from the DNA-BD to theADvectorandviceversa.
Remove the activating domain bycreatingspecificdeletionswithinthegene.Retestthedeletionconstructsforactivation.Attheaminoacidlevel,anetnegativechargeper10aminoacids is a minimal AD. Note thatsuchdeletionsmayalsoeliminateapotentiallyinteractingdomain.
Remake SD/–Ade/–His/–Leu/–Trp/X-α-Galmedium.Add theappropriateamountof3-AT(SectionV.B.5).
UsewaterorTE.
Remake media, test with controltransformations.
Switchtosequentialtransformation.
Repeat the experiment using moreof the plasmid that had the lowtransformationefficiency.
Check the purity of the DNA and,if necessary, repurify it by ethanolprecipitation.
If you are not already doing so,we strongly recommend using thepretestedandoptimizedYeastmakerCarrier DNA, which is availableseparately(Cat.No.630440),oraspartoftheYeastmakerYeastTransformationSystem(Cat.No.630439).
Repeatthetransformation,thistimeincluding a “recovery” period aftertheheatshock.Toprovidearecoveryperiod, perform the simultaneouscotransformation as described(SectionIX.B),butaddthefollowingstepsafterStepB.21:22. Resuspendcellsin1.0LofYPDA
medium for a library-scale,and 100 ml for a large-scale,transformation.
23. Incubate cells for 1 hr at 30°Cwithshakingat230rpm.
24. Pellet cells by centrifuging at1,000 x g for 5 min at roomt e m p e r a t u r e . R e m o v es u p e r n a t a n t . P r o c e e dtoStepB.22.
Thebaitproteinhasatranscriptionalactivationdomain.Thisisespeciallylikely if the bait protein is atranscription factor (Ma & Ptashne,1987; Ruden et al., 1991; Ruden,1992). Acidic amphipathic domainsare often responsible for unwantedtranscriptional activation (Ruden et al.,1991;Ruden,1992).
Impropermediapreparation.
ResuspensionoftransformedcellsinYPDAistoorich.
Impropermediapreparation.
A problem with simultaneouscotrans-formation, even though thetransformation with the AD libraryplasmidsalonegaveatransformationefficiencyof≥5x104cfu/µgandwiththe bait plasmid alone gave ≥105cfu/µg.
The AD library vector gave atransformationefficiencyof<5x104cfu/µg or the bait plasmid gave atransformationefficiencyof<105forthebaitplasmid.
Problem Cause Solution
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XI.TroubleshootingGuidecontinued
Comparegrowthcurvesofhoststrainwith DNA-BD vector and DNA-BD/bait. If bait is toxic, use sequentialtransformations or switch to a lowexpressingDNA-BDvector.
Truncationofoneofthehybridproteinsmay alleviate the toxicity and stillallowtheinteractiontooccur.Tryusingvectors that express lower levels ofthefusionproteins,suchaspGBT9(aDNA-BDvector),andpGAD424,pGADGL,orpGAD10(ADvectors)(Holtz&Zhu,1995).
See previous tip on improvingtransformationefficiency.
Constructhybridscontainingdifferentdomains of the bait protein. Forexample, to study proteins thatnormallydonotlocalizetothenucleus,it may be necessary to generatemutantformsoftheproteinthatcanbe transported across the nuclearmembrane.
UsetheMatchmakerLexATwo-HybridSystem(Cat.No.K1609-1).
RefertoSectionXformethodstoverifyprotein interactions; see Bartel et al. (1993a) for further discussion of falsepositives.
In sequential transformation, ADtransforms poorly; even as emptyvector.Baitprotein ismildlytoxicorinhibitingtotransformation
High-levelexpressionofoneorbothof thehybridproteins is toxic to thecell;therefore,transformantswillnotgroworwillgrowveryslowly.Forthisreason,werecommendthatyoucheckfor cell toxicity before performing alibraryscreen(SectionVIII.D).
The transformationefficiencyofoneorbothplasmidsistoolow.Youmaynotbescreeningasufficientnumberof library cotransformants.This canbecritical,especiallyiftheinteractingtarget protein is encoded by a raretranscriptinthesourcetissue.
If one of the following situations isoccurring, it may interfere with theability of the two hybrid proteinsto interact: (1) the hybrid proteinsarenot stablyexpressed in thehostcell; (2) the fused GAL4 domainsoccludethesiteofinteraction;(3)thehybrid protein folds improperly; or(4) the hybrid protein cannot belocalizedtotheyeastnucleus.(SeevanAelstet al.[1993]foroneexample.)
Some types of protein interactionsmaynotbedetectableinaGAL4-basedsystem.
Some protein interactions are notdetectableusinganytypeoftwo-hybridassay.
A rare category of false positives inwhich anAD/library hybrid activatestranscriptioninappropriately.
Failuretodetectknownproteininteractions
AD/libraryplasmidactivatesallthreereportersindependentoftheDNA-BD/bait
Problem Cause Solution
Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3247-1 30 Version No. PR742219
XII.References
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Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3247-1 32 Version No. PR742219
XIII.RelatedProducts
ForthelatestandmostcompletelistingofallClontechproducts,pleasevisitwww.clontech.com
GAL4-basedOne-andTwo-HybridSystemsandRelatedProducts: • pGADT7ADVector 630442 • pGBKT7DNA-BDVector 630443 • MammalianMatchmaker™Two-HybridAssayKit 630301 • Matchmaker™PretransformedcDNALibraries many • Matchmaker™cDNAandGenomicLibraries many • Matchmaker™RandomPeptideLibrary 638853 • pBridgeVector 630404 • pCMV-Myc&pCMV-HAVectorSet 631604 • Matchmaker™Co-IPKit 630449 • Matchmaker™ADLD-InsertScreeningAmplimerSet 630433
Antibodies: • c-MycMonoclonalAntibody 631206 • c-MycMonoclonalAntibody-AgaroseBeads 631208 • HA-TagPolyclonalAntibody(AffinityPurified) 631207 • GAL4ADMonoclonalAntibody 630402 • GAL4DNA-BDMonoclonalAntibody 630403 GeneralReagentsforWorkingWithYeast: • Yeastmaker™YeastTransformationSystem2 630439 • Yeastmaker™CarrierDNA 630440 • Yeastmaker™YeastPlasmidIsolationKit 630441 • KC8ElectrocompetentandChemicallyCompetentCells 630435 630434 • YPDMedium 630409 • YPDAgarMedium 630410 • MinimalSDBase(containsglucoseorgalactose) 630411 630420 • MinimalSDAgarBase(containsglucoseorgalactose) 630412 630421 • DOSupplements many • X-α-Gal 630407
GeneralCloningReagents: • Advantage®2PolymeraseMix 639201 639202 • Advantage®2PCRKit 639206 639207 • NucleoSpin®ExtractionKit 635961
Protocol No. PT3247-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR742219 33
Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual
AppendixA.Media&SolutionRecipes
MediaforGrowthandSelectionofYeastClontechcarriesafulllineofyeastmediaincludingYPD,SDwithglucoseorgalactose;withorwithoutagar,andDropout(DO)SupplementsidealforusewithMatchmakerTwo-HybridSystemsandLibraries.PleaseseeSectionXIIIfororderinginformation.IfyoupurchasedyeastmediafromClontech,followthedirectionsprovidedwiththeproduct.Alternatively,youcanprepareyourownmediaandDOSupplementsusingthedetailedrecipesprovidedintheYPH,AppendixC.
• YPDAmedium
To1LofYPDMedium(Cat.No.630409),add15mloffilter-sterilized0.2%adeninehemis-ulfate(SigmaCat.No.A-9126)toafinalconcentrationof0.003%.
Reagentforlowstringencyscreens
• 65%glycerol/MgSO4solution(sterile)
FinalConc.
Glycerol 65%v/v
MgSO4 100mM
Tris-HCl(pH8.0) 25mM
MediaforTiteringandAmplifyingPlasmidLibrariesinE. coli • LBbroth
10g/L Bacto-tryptone
5g/L Bacto-yeastextract
5g/L NaCl
AdjustpHto7.0with5NNaOH.Autoclave.Storeatroomtemperature.
• LB/ampbroth
PrepareLBbroth,thenautoclaveandcoolto50°C.Addampicillinto100µg/ml.Storeat4°C.
• LB/ampplates
PrepareLBbroth, thenaddagar (18g/L),autoclave,andcool to50°C.Addampicillin to100µg/ml.Pourplatesandstoreat4°C.
• Ampicillinstocksolution(50mg/mlinH2O;1000X).Storeat–20°C.
X-α-Gal
DissolveX-α-Galat20mg/mlindimethylformamide(DMF).StoreX-α-Galsolutionsinglassorpolypropylenebottlesat–20°Cinthedark.
1.PouringX-α-Galindicatorplates a. Prepareandautoclave1.0Loftheappropriatedropoutagarmedium.Coolto55°C. b. Add1mlofX-α-Gal(20mg/ml). c.Pourplatesandallowmediumtohardenatroomtemperature. d. Platecellsandincubateattheappropriatetemperatureuntilbluecoloniesform. 2.SpreadingX-α-Galontopremadeplates a. DiluteX-α-Galto4mg/mlinDMF. b.Pourappropriatedropoutplatesandallowmediumtohardenat
roomtemperature.
c. Spread200µlofX-α-Gal(4mg/ml)ontoa15-cmplateor100µlontoa10-cmplateusingglassbeads.
Note:Toquickly(1–24hr)determineifayeaststraincontainsMEL1,spreadX-α-Galat20 mg/mlasdescribed.
d. Allowplatestodryfor15minatroomtemperature. e. Platecellsandincubateattheappropriatetemperatureuntilbluecoloniesform.
Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3247-1 34 Version No. PR742219
A. Important: • Dilutedlibrariesarealwayslessstablethanundilutedlibraries.Therefore,oncethelibrary
dilutionsaremade,usethemwithinthenexthourbeforedrasticreductionsintiteroccur. • Usepropersteriletechniquewhenaliquotingandhandlinglibraries. • Designanduseappropriatecontrolstotestforcross-contamination. • Always use the recommended concentration of antibiotic in the medium to ensure
plasmidstability. • pACTandpACT2librariesarereleasedfromλACTandλACT2libraries,respectively.Incubating
culturesofpACTandpACT2librariesat37°Ccanresultinplaquesonthehigh-densityplatesduetothepresenceofresidualphageinthelibrary.Theseplaquesshouldnotinterferewithlibrarytitering.However,iftheyarenumerous,retiterthelibraryat30–31°Candincubate36–48hr.
B. PlasmidLibraryTitering
ReagentsandMaterialsRequired:
• LBbroth(AppendixA) • LB/ampplates(100-mmplates;AppendixA) Note:Allowtheagarplatestodryunsleevedatroomtemperaturefor2–3days,orat30°Cfor3hr,priorto
platingcells.Moisturedropletsontheagarsurfacecanleadtounevenspreadingofcells.
• Sterileglassspreadingrod,bentPasteurpipette,or5-mmglassbeadsforspreadingcultureonplates.
1.Thawanaliquotofthelibrary,andplaceonice. 2.Mixbygentlevortexing.Transfer1µlto1mlofLBbrothina1.5-mlmicrocentrifugetube.
Mixbygentlevortexing.ThisisDilutionA(1:103). Note:pACTandpACT2librariesmaybeviscous,andrepeatedfreeze/thawcyclesmayincreaseviscosity.To
facilitateaccuratepipettingofthelibrary,firstdilutea10-µlsamplewith10µlofLBbroth.Thenpreparefurtherdilutionsfromthis1:1dilution.Besuretoaccountforthisextradilutionwhencalculatingthetiter.
3.Remove1µlfromDilutionA,andadditto1mlofLBbrothina1.5-mlmicrocentrifugetube.Mixbygentlevortexing.ThisisDilutionB(1:106).
4.Add1µlfromDilutionAto50µlofLBbrothina1.5-mlmicrocentrifugetube.Mixbygentlevortexing.SpreadtheentiremixtureontoaprewarmedLB/ampplate.
Note:Continuespreadingtheinoculumovertheagarsurfaceuntilallvisibleliquidhasbeenabsorbed.Thisprocedureisessentialforevengrowthofthecolonies.
5.Plate50-µland100-µlaliquotsofDilutionBonLB/ampplates. 6.Leaveplatesatroomtemperaturefor15–20mintoallowtheinoculumtosoakintotheagar. 7.Inverttheplates,andincubateat37°Cfor18–20hr,orat30–31°Cfor24–36hr. • ForpACTandpACT2libraries:incubateplatesat30–31°Cfor36–48hr. 8.Countthenumberofcoloniestodeterminethetiter(cfu/ml).Calculatethetiterasfollows: • DilutionA:No.coloniesx103x103=cfu/ml • DilutionB:(No.colonies/platingvolume)x103x103x103=cfu/ml Note:A2–5-foldrangeintitercalculationsisnotunusual,especiallyifmorethanonepersonisdoingthetitering.
AppendixB.LibraryTitering
Protocol No. PT3247-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR742219 3�
Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual
YoumustamplifypremadeMatchmakerLibrariestoobtainenoughplasmidforlibraryscreeninginyeast.Youwillneed100–500µgofplasmidDNAtoscreen~1x106independentclones(seeTableIV).
Note:pACTandpACT2librarycustomers:Ifyouobservedplaquesonyourhigh-densitylibrarytiteringplates(AppendixB),youmaywishtousealowertemperature(i.e.,30–31°C)whenincubatingthelibraryamplificationplates.Thelowertemperaturewillrequirealongerincubationtime,asnotedbelow.IfyoufollowthisamplificationprotocolexactlythroughStep7,afewλplaquesontheamplificationplatesshouldnotaffectthequalityoryieldofplasmid.IfyouchoosetoincludeStep8,besuretoperformitat30°C.Lysisismorelikelytooccurinliquidcultures,andyourisklysingtheentirecultureattemperaturesover31.0°C.Growthat30–31°CisnotnecessaryoncethepACTorpACT2libraryDNAhasbeentransferredtoanewE. coli host.
A. ReagentsandMaterialsRequired • LB/ampagarplates(AppendixA)
Notes:• Theexactnumberofplatesrequireddependsonthesizeofthelibrary.Usethefollowingcalculationtodetermine
howmanyplatestouse.Normally,use3timesthesizeoftheoriginallylibraryandplateat20,000cfu/plate. (No.ofindependentclonesx3)=No.clonestoscreen 2x106x3=6x106clonestoscreen No.ofclonestoscreen/coloniesperplate=No.ofplates 6x106/20,000=300plates • Ifthetiteris6x108,determinetheamountofthelibrarystocktospreadoneachplate. No.clonestoscreen/librarytiter=μlsoflibrarytoplate 6x106/6x108titer=10μl • Calculatethevolumeofmedianeededtoplate150μloneachplate. 300platesx150μl=52.5ml • Add10μlofthelibraryto52.5mlofLBampandspread150 μlontoeachofthe300LBampplates • Allowtheagarplatestodryatroomtemperaturefor2–3days,orat30°Cfor3hr,priortoplatingcells.
Moisturedropletsontheagarsurfacecanleadtounevenspreadingofcells.
• LB/glycerol(1L;LBbrothcontaining25%glycerol) • Sterileglassspreadingrod,bentPasteurpipette,or5mmdiametersterileglassbeads(~10/
plate) • [Optional,forStep8]LB/ampbroth(2L;AppendixA)andsterile,50–80%glycerol.
B. PlasmidLibraryAmplificationProtocol 1.Ifyouhavenotdonesoalready,titertheplasmidlibrary(AppendixB). 2.PlatethelibrarydirectlyonLB/ampplatesatahighenoughdensitysothattheresulting
colonieswillbenearlyconfluent(~20,000–40,000cfuper150-mmplate).Plateenoughcfutoobtainatleast2–3Xthenumberofindependentclonesinthelibrary.
Notes:
• Thenumberofindependentclonesisthenumberofindependentcoloniespresentinthelibrarybeforeamplification.IfyouhavepurchasedalibraryfromClontech,thesizeofthelibraryisstatedonthePAC.
• Topromoteevengrowthofthecolonies,continuespreadingtheinoculumovertheagarsurfaceuntilallvisibleliquidhasbeenabsorbed,andthenallowplatestositatroomtemperaturefor15–20min.Ifusingglassbeadstospreadthecolonies,shaketheplatebackandforth—notroundandround.
3.Inverttheplates,andincubateat37°Cfor18–20hr. Notes:
• ForpACTorpACT2libraries:incubateplatesat30–31°Cfor36–48hr,oruntilconfluent. • Growingthetransformantsonsolidmediuminsteadofinliquidcultureminimizesunevenamplification
oftheindividualclones.
4.Add ~5 ml of LB/glycerol to each plate and scrape colonies into liquid. Pool all theresuspendedcoloniesinoneflaskandmixthoroughly.
Note:Toobtainhigheryields,scrapethecoloniesintoLB/amp(noglycerol)andpoolthecoloniesinoneflask.Incubateat30–31°Cfor2–4hrwithvigorousshaking(200rpm).Addsterileglycerolto25%,andproceedtoStep5.
AppendixC.PlasmidLibraryAmplification
Matchmaker™ GAL4 Two-Hybrid System 3 & Libraries User Manual
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3247-1 3� Version No. PR742219
5.Setasideone-thirdofthelibraryculture(roughlyequivalentto3Lofovernightculture)fortheplasmidpreparation;thisportioncanbestoredat4°Cifyouplantouseitwithin2weeks.Forstorage>2weeks,dividethecultureinto50-mlaliquots,andstoreat–70°C.
• Setasidefive1-mlaliquotsofthelibrarycultureincaseyouwishtore-amplifythelibraryatalatertime.Storethealiquotsat–70°C.
• Dividetheremainderofthelibrarycultureinto50-mlaliquotsandstoreat–70°C. 6.PrepareplasmidDNAusinganystandardmethodthatyieldsalargequantityofhighly
purifiedplasmid.(SeeSambrooketal.,1989forCsClgradientpurification,ifnecessary.)Note:ThecellculturefromStep4willbeverydense(OD600>>1),soadjusttheplasmidpreparationprotocolaccordingly(i.e.,followtheprocedureasifyouwereprocessing3Lofovernightliquidculture)orprepareasteriledilutionfrom10-1to10-3.PlasmidpreparationproceduresarebasedonacultureofOD600=1–2.Yourplasmid preparation must take into account the much higher than normal cellular concentration and bemagnifiedaccordingly.If30mlofculturewhendilutedto10-2hasanOD600=1,treatthestockasthoughitwere3LofcultureandprepareplasmidDNAusingaNucleoBondMegaorGigaPlasmidKit(Cat.Nos.635938&635939).
7.ExpectedyieldsofplasmidDNAper1x106cfu: • pACT&pACT2Libraries:0.25mg • allotherMatchmakerGAL4Libraries:~1mg 8.[Optional]Toobtainhigheryields,scrapethecolonies intoLB/amp(noglycerol)and
poolthecoloniesinoneflask.Incubateat31–30°Cfor2–4hrwithat200rpm.Addsterileglycerolto25%,thenproceedwithStep5above.
AppendixC.PlasmidLibraryAmplificationcontinued