mTn-4xHA/lacZ

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

Inmoredetail:mTn-4xHA/lacZcaneasilybeinsertedatmutiplesitesinagivengene.ThemutagenizedDNAisthentransformedintoyeast,whereitreplacesthechromosomallocusbyhomologousrecombination.Thetransposoninsertionscreateapoolofinsertion/disruptionalleles.Insertionsthatgeneratein-framefusionofthecodingregiontolacZcanbeusedtomonitorandquantifygeneexpression,viaassaysforbeta-galactivity.ThetransposoncanalsobeexcizedbyCre-mediatedrecombinationtoleavea5base-pairduplicationcausedbytransposoninsertionplusa262-bpinsertioncontainingsequencesencoding4copiesoftheHAepitope.WhenlacZisfusedin-frametothegeneofinterest,theexcisioneventresultsinanin-frameinsertionof89aminoacids,calledaHATtag,intotheencodedprotein.InsertionoftheHATtaghasthepotentialtocreateconditionally-defectiveformsoftheprotein.

TheaccessionformTn-4xHA/lacZisU54829.

AkitformutagenesisofayeastgenewithmTn-4xHA/lacZisavailable.

• Seewhatitcontains
• Orderthekit.

Pleasereadthiswholedocumentbeforeyoustart!

Shuttlemutagenesis

• ClonefragmentintovectorpHSS6.
  • pHSS6isfromstrainR1123;mapgivenbelow.
  • DeleteasmuchofthepolylinkeraspossIBLeassometimestransposon"hot-spots"intoit.
  • SelecttransformantsonLBKan40.
• TransformthisplasmidintocompetentcellsofR1236/B211.
  • SelectonLBKan40Cm34.
• TransferF::mTn-4xHA/lacZintocellsbymatingwithstrain#94.
  • Growstrainsovernightwithantibioticselection(Tet3for#94).
  • Subculture1:100infreshmedium(noantibiotics).Growat37^oCtoearlylogphase(whencellswirlsarevisible).Therecipientstrain(B211)canbedenserthanthedonor.
  • Mix200ulofeachstrain.Incubateat37^oCwithoutagitationfor20minto1hr.
  • Plateas100ulaliquotsontoLBTet3Kan40Cm34.
  • DotheControl:Spotthestartingstrainsontothismedia.
  • Grow1-2daysat30^oC.Nowyouhavecointegrates.
  • Setupstrain#70inSm50Cm34overnight.
• Matetostrain#70toresolvecointegrates
  • Elutecoloniesfromplates:put2mlsofLBontheplate,scrapeoffthecolonieswithaspeader.Thisisyoureluate.Youshouldhaveseveralthousandcoloniesatleast.
  • Diluteovernightcultureofstrain#701:100withoutantibiotic.Diluteeluatetoroughlysamedensity.
  • Growandmateasbefore.
  • Aftermatingfor20minto1hr,plate100ulaliquotsonLBTet3Kan40Sm50andgrowovernightat37^oC.
  • DotheControl:Spotthestartingstrainsontothismedia.
• RescueresolvedDNAfromthisstrain
  • EluteyourcoloniesoffinLB.Again,youshouldhavethousands.DilutesomeeluateinLBTet3Kan40togiveanalmostsaturateddensity.Growat37^oCforafewhours.
  • IsolateDNAbyminiprep.(Wedoastandard1-2-3alkalinelysisbutuse150ulof7.5MNH4AcassolutionIII,and270ulofisopropanoltoprecipitate.Thisremovesmostofprotein(avoidingphenol)andRNA,givingaverysmallcleanpellet.Still,therearenucleasessowekeepeverythingonice).
  • Transformabout1/10ofminprepintoaregularrecAendAcloningstrain(egDH5).PlateonLBTet3Kan40.
• Transformintoyeast.
  • Eluteentirepooloftransformants(again,aimforthousands)andmakeaminiprepasinstep5.(Make-70^oCstockofbacterialpoolforfutureuse).
  • TransformNotIdigestofentirepoolintoyeast,selectingforURA3.LookforlacZfusionsamongtransformants.
  • N.B.ForHATepitope-tagging,youmaywanttopre-transformyouryeaststrainwithpB227/GAL-cre(selectingLEU2).

Screeningforin-framelacZfusionsinyeast

1. TransformantcoloniesarepatchedtoSC-ura(SC-ura-leuifyoualreadyhavepB227/GAL-creinthere).
2. CellsarereplicaplatedtoanSC-ura(-leu)plateandaSC-uraplateonwhichasterilediscofWhatman1Afilterpaperhasbeenplaced,andgrownovernightat30^oC.Othermediaorgrowthconditionscanbesubstitutedasdesired.Forade2strains,anytestmediashouldcontain80mg/lofadenine,astheredpigmentcanobscuretheX-galresult.
3. Filtersareliftedfromtheplatesandplacedinthelidofa9-cmglasspetridish.Thislidisthenplacedinsideaclosed15-cmglasspetridishcontainingchloroform,for10to30minutes.Theminimumexposuretimenecessaryforaparticularyeaststraincanbedeterminedempirically.
4. Filtersareplacedcolony-sideupontoX-Galplates(120ug/ml5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside,0.1MNaPO4[pH7]and1mMMgSO4in1.6%agar)andincubatedat30^oCforupto2days.
5. TransformantscarryingproductivelacZfusionsarerecoveredfromtheregrownSC-ura(-leu)plate.ItisadvisabletosubsequentlymaintainselectionforURA3whereverpossible,assomemutationsaredeleteriousevenintheheterozygousstate.
6. PCRprimersdesignedusingthemTn-3xHA/lacZsequencecanbeusedtodeterminepositionofthetransposon.TheIRelementsandpalindromicloxregionsshouldbeavoided.

UsingtheexcisionfeaturetoHAT-epitopetagaprotein

Aleu2ura3GAL+yeaststrainisrequired.Whentransposoninsertionhascreatedanin-framefusiontolacZinthegeneofinterest,thetransposoncanbeexcizedbyCre-mediatedrecombinationtoleavea262-bpinsertion(sequencegivenbelow)containingfourcopiesoftheHAepitope.Withthe5basepairduplicationcausedbytransposoninsertion,thisgivesanin-frame89aminoacidinsertion.ThepopouteventismediatedbycrerecombinaseandrequiresinductionoftheGAL1-10promoterongalactose.Ourstrainsgrowpoorlyongalactosebutgive80to100%popouts.

TheHAtagcanbedetectedbymousemonoclonalantibodies12CA5(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.
   • Spota10uldropontoanFOAplateandstreakitforsinglecolonies(non-quantitativeapproach!).
   • Alternatively,platedilutionsontoSCmediaandreplicatoSC-uratoidentifyUra-colonies.Theinducedculturesshouldgive100-foldmoreUra-cellsthanthecontrol.
5. PCRprimersdesignedusingthesequencegivenbelowcanbeusedtodeterminepositionofthetag.TheTRelementsandpalindromicloxRregionshouldbeavoided.

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

SequenceofHATtag(4xHA):

TRinuppercase.loxRinbold.

GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGgcggcctacccatacgacgtcccagactacgcgttggccgctatcgcttcggataactcctgctatacgaagttatgggcggccgtttacccatacgatgttcctgactatgcgggctATCCctatgacgtcccggactatgcaggatcctatccatatgacgttccagattacgctccggccgcCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC

Bacterialstrainsused(providedinkit):

Vectorused:

TheaccessionforpHSS6isM84115.

Antibioticsused:

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