mTn-3xHA/GFP

Uses:Genedisruption,analysisofgeneexpression,creatingfusiontoGFP,HATepitope-taggingproteinatrangeofsites,creatingconditionalalleles.

Inmoredetail:mTn-3xHA/GFPcanbeusedeasilytocreatealibraryofinsertions,eachatadifferentsiteinagivengene.ThemutagenizedDNAisthentransformedintoyeast,whereitreplacesthechromosomallocusbyhomologousrecombination.Thetransposoninsertionscreateapoolofinsertion/disruptionalleles.Insertionsthatgeneratein-framefusionofthecodingregiontoGFPcanbeusedtomonitorandquantifygeneexpression,viaassaysforfluorescenceactivity.LocalizationoftheGFPfusionproteincanbeexaminedbyfluorescencemicroscopy.ThetransposoncanalsobeexcizedbyCre-mediatedrecombinationtoleavea5base-pairduplicationcausedbytransposoninsertionplusa274-bpinsertioncontainingsequencesencodingthe3xHAtagandthefactorXaproteasecleavagerecognitionsite.WhenGFPisfusedin-frametothegeneofinterest,theexcisioneventresultsinanin-frameinsertionof93aminoacids,calledaHATtag,intotheencodedprotein.TheHATtagallowsimmunodetectionoftheprotein.InsertionoftheHATtagalsohasthepotentialtocreateconditionally-defectiveformsoftheprotein.

TheaccessionnumberformTn-3xHA/GFPisU54830.

AkitformutagenesisofayeastgenewithmTn-3xHA/GFPisavailable.

• Seewhatthekitcontains
• Orderthekit

Pleasereadthiswholedocumentbeforeyoustart!

Shuttlemutagenesis

1. ClonetargetgeneintovectorpHSS6.
   • pHSS6isinstrainR1123;mapgivenbelow.
   • DeleteasmuchofthepolylinkeraspossIBLeassometimestransposon"hot-spots"intoit.
   • SelecttransformantsonLBKan40.
2. TransformtargetplasmidintocompetentcellsofR1236/B211.
   • SelectonLBKan40Cm34.
3. TransferF::mTn-3xHA/lacZintocellsbymatingwithstrain#111/B428.
   • Growstrainsovernightwithantibioticselection(Tet3for#111/B428).
   • Subculture1:100infreshLBmedium(noantibiotics).Growat37^oCtoearlylogphase(whencellswirlsarevisible).Therecipientstrain(R1236/B211)canbedenserthanthedonor(#111/B428).
   • Mix200ulofeachstrain.Incubateat37^oCwithoutagitationfor20minto1hr.
   • Plateas100ulaliquotsontoLBTet3Kan40Cm34.DotheControl:Spotthestartingstrainsontothismedia.
   • Grow1-2daysat30^oC.Nowyouhavecointegrates.
   • Setupstrain#70/B425inSm50Cm34overnight.
4. Resolvecointegratesbymatingtostrain#70/B425.
   • Elutecoloniesfromplates:put2mlsofLBontheplate,scrapeoffthecolonieswithaspeader.Thisisyoureluate.Youshouldhaveseveralthousandcoloniesatleast.
   • Diluteovernightcultureofstrain#70/B4251:100withoutantibiotic.Diluteeluatetoroughlysamedensity.Growandmateasbefore.
   • Aftermatingfor20minto1hr,plate100ulaliquotsonLBTet3Kan40Sm50andgrowovernightat37^oC
   • DotheControl:Spotthestartingstrainsontothismedia.
5. RescueresolvedDNAfromthisstrain.
   • EluteyourcoloniesoffinLB.Again,youshouldhavethousands.DilutesomeeluateinLBTet3Kan40togiveanalmostsaturateddensity.Growat37^oCforafewhours.
   • IsolateDNAbyminiprep.(Wedoastandard1-2-3alkalinelysisbutuse150ulof7.5MNH4AcassolutionIII,and270ulofisopropanoltoprecipitate.Thisremovesmostofprotein(avoidingphenol)andRNA,givingaverysmallcleanpellet.Still,therearenucleasessowekeepeverythingonice).
   • Transformabout1/10ofminprepintoaregularrecAendAcloningstrain(egDH5).PlateonLBTet3Kan40
6. Transformintoyeast.
   • Eluteentirepooloftransformants(again,aimforthousands)andmakeaminiprepasinstep5.(Make-70stockofbacterialpoolforfutureuse).
   • TransformNotIdigestofentirepoolintoyeast,selectingforURA3.
   • NBForHATepitope-tagging,youmaywanttopre-transformyouryeaststrainwithpB227/GAL-cre(selectingLEU2)

Screeningforin-frameGFPfusionsinyeast

WehavenotdoneassaysofGFPactivityinyeast.

SeeNiedenthaletal(1996)fortheirmethods.

AnalyzingGFPfusionproteinlocalizationinyeast

WetestedmTn-3xHA/GFPbymutagenesisoftheBDF1gene,whichencodesachromatin-associatedprotein.Wegrewindividualbdf1::mTn-3xHA/GFPtransformantstoadensityof10⁷cells/mlinSC-ura.Thelastfourhoursofgrowthwereatroomtemperature,toallowformationoftheGFPchromophore.ThenweexaminedcellsdirectlyusingaLeitzmicroscopywithasystem13filter(thismaynotbeoptimal).In4of38transformants,wesawgreenfluorescenceofthenucleus.Fixationandspheroplastingofthecellsimprovedthesignal-to-noiseratio.

UsingtheexisionfeaturetoHAT-epitopetagaprotein

Aleu2ura3GAL+yeaststrainisrequired.Whentransposoninsertionhascreatedanin-framefusiontoGFPinthegeneofinterest,thetransposoncanbeexcizedbyCre-mediatedrecombinationtoleavea274bpinsertion(sequencegivenbelow)containingthe3xHAtag.Withthe5basepairduplicationcausedbytransposoninsertion,thisgivesanin-frame93aminoacidinsertion.ThepopouteventismediatedbycrerecombinaseandrequiresinductionoftheGAL1-10promoterongalactose.Ourstrainsgrowpoorlyongalactosebutgive80to100%popouts.

TheHAtripletagcanbedetectedbymousemonoclonalantibodies12CA5(Boehringer)orMMS101R(BAbCo,Richmond,California).Theseantibodyrecognisecross-reactingyeastproteinsofabout55kDor110kD,respectively,andcangiveaspottybackgroundonimmunofluorescence.Despitethisdrawback,the3xHAtaghasbeenusedextensivelyandsuccessfullyinyeast.Arabbitpolyclonalantiseraisalsoavailable(101c500;BabCo)butthiswaslessreactiveintheoneinstancewetried.Protocolsforyeastimmunofluorescencecanbefoundhere,orinMethodsinEnzymology194(1991).

1. TransformstrainwithplasmidpB227/GAL-cre,selectingonSC-leu.
2. Inoculatetransformantsinto2mlsSC-ura-leuwith2%raffinoseascarbonsource,andgrowtosaturation.
3. Dilute1/100intoSC-leuwith2%galactoseascarbonsource.Asacontrolalsodilute1/100intoSC-leuwith2%glucoseascarbonsource.Growfor2days(somestrainsinducewithoutgrowing).
4. Ifgrown,dilute1/100.Otherwise,proceedwithundilutedculture.
5. Spota10uldropontoanFOAplateandstreakitforsinglecolonies(non-quantitativeapproach!).Alternatively,platedilutionsontoSCmediaandreplicatoSC-uratoidentifyUra-colonies.Theinducedculturesshouldgive100xmoreUra-cellsthanthecontrol.
6. PCRprimersdesignedusingthesequencegivenbelowcanbeusedtodeterminepositionofthetag.TheIRelementsandpalindromicloxRregionshouldbeavoided.

N.B.Whentaggingessentialgenes,theoriginalstraintransformedshouldobviouslybediploid.YoucandissecttheHAT-taggedversiontoseeifthetaggedgeneisfunctional.Toberigorous,onlybelieveatagislethalifitiscomplementedbythewild-typegene,andifseveralpopouteventsgivethesamephenotype.

SequenceofHATtag(3xHA):

TRinuppercase.loxRinbold.

GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGgcggccattgaaggtagaagagaaAATttgtacttccaaagaaagaaggccgctatcgcttcggataactcctgctatacgaagttatgggcggccgtttacccatacgatgttcctgactatgcgggctatccctatgacgtcccggactatgcaggATCCtatccatatgacgttccagattacgctccggccgcCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC

Bacterialstrainsused(providedinkit):

Vectorused:

TheaccessionforpHSS6isM84115

Antibioticsused:

NB.Whenonlyafewplatesofeachtypeareused,it"sconvenienttochopanLBplateupwithasteriletoothpick,putthebitsinasterileflask,andmelttheagarbymicrowave.Addappropriateamountsofantibioticandrepourplates.