mTn-3xHA/lacZ

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

Inmoredetail:mTn-3xHA/lacZcaneasilybeinsertedatmutiplesitesinagivengene.ThemutagenizedDNAisthentransformedintoyeast,whereitreplacesthechromosomallocusbyhomologousrecombination.Thetransposoninsertionscreateapoolofinsertion/disruptionalleles.Insertionsthatgeneratein-framefusionofthecodingregiontolacZcanbeusedtomonitorandquantifygeneexpression,viaassaysforbeta-galactivity.ThetransposoncanalsobeexcizedbyCre-mediatedrecombinationtoleavea5base-pairduplicationcausedbytransposoninsertionplusa274-bpinsertioncontainingsequencesencodingthe3xHAtagandthefactorXaproteasecleavagerecognitionsite.WhenlacZisfusedin-frametothegeneofinterest,theexcisioneventresultsinanin-frameinsertionof93aminoacids,calledaHATtag,intotheencodedprotein.InsertionoftheHATtaghasthepotentialtocreateconditionally-defectiveformsoftheprotein.

GenBankAccession:U54828.

AkitformutagenesisofayeastgenewithmTn-3xHA/lacZisavailable.

• Seewhatitcontains
• Orderthekit

Pleasereadthiswholedocumentbeforeyoustart

Shuttlemutagenesis

1. ClonefragmentintovectorpHSS6.
   • pHSS6isfromstrainR1123;mapgivenbelow.
   • DeleteasmuchofthepolylinkeraspossIBLeassometimestransposon"hot-spots"intoit.
   • SelecttransformantsonLBKan40.

2. TransformthisplasmidintocompetentcellsofR1236/B211.

   • SelectonLBKan40Cm34.
3. TransferF::mTn-3xHA/lacZintocellsbymatingwithstrain#95.
   • Growstrainsovernightwithantibioticselection(Tet3for#95).
   • Subculture1:100infreshmedium(noantibiotics).Growat37^oCtoearlylogphase(whencellswirlsarevisible).Therecipientstrain(B211)canbedenserthanthedonor.
   • Mix200ulofeachstrain.Incubateat37^oCwithoutagitationfor20minto1hr.
   • Plateas100ulaliquotsontoLBTet3Kan40Cm34.
   • DotheControl:Spotthestartingstrainsontothismedia.
   • Grow1-2daysat30^oC.Nowyouhavecointegrates.
   • Setupstrain#70inSm50Cm34overnight.
4. Matetostrain#70toresolvecointegrates
   • Elutecoloniesfromplates:put2mlsofLBontheplate,scrapeoffthecolonieswithaspeader.Thisisyoureluate.Youshouldhaveseveralthousandcoloniesatleast.
   • Diluteovernightcultureofstrain#701:100withoutantibiotic.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..LookforlacZfusionsamongtransformants.
   • 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.

UsingtheexisionfeaturetoHAT-epitopetagaprotein

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

TheHAtripletagcanbedetectedbymousemonoclonalantibodies12CA5(Boehringer)orMMS101R(BAbCo,Richmond,California).Theseantibodyrecognisecross-reactingyeastproteinsofabout55kDor110kD,respectively,andcangiveaspottybackgroundonimmunofluorescence.Minimizingthedegreeofspheroplastingreducesbackgroundconsiderably.Despitethisdrawback,the3xHAtaghasbeenusedextensivelyandsuccessfullyinyeast.Arabbitpolyclonalantiseraisalsoavailable(101c500;BabCo)butthiswaslessreactiveintheoneinstancewetried.Protocolsforyeastimmunofluorescencecanbefoundelswhereonthiswebsite,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.Toberigorous,onlybelieveatagislethalifitiscomplementedbythewild-typegene,andifseveralpopouteventsgivethesamephenotype.

SequenceofHATtag(3xHA):

38bpTerminalRepeatinuppercase.loxRinbold.

GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGgcggccattgaaggtagaagagaaAATttgtacttccaaagaaagaaggccgctatcgcttcggataactcctgctatacgaagttatgggcggccgtttacccatacgatgttcctgactatgcgggctatccctatgacgtcccggactatgcaggATCCtatccatatgacgttccagattacgctccggccgccCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC

Bacterialstrainsused(providedinkit):

Vectorused:

TheaccessionforpHSS6isM84115.

Antibioticsused:

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