mTn-3xHA/lacZ

TR	Tn3terminalinvertedrepeats
Xa	FactorXacleavagerecognitionsite
loxR	loxsite,targetforCrerecombinase
lacZ	5"-truncatedlacZgeneencodingbeta-galactosidase
URA3	URA3genefromS.cerevisiae
tet	Tetracyclineresistancegene
res	Tn3siteforresolutionoftranspositionintermediate
loxP	loxsite,targetforCrerecombinase
3xHA	Hemagglutinin(HA)tripleepitopetag

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

ClonefragmentintovectorpHSS6.
- pHSS6isfromstrainR1123;mapgivenbelow.
- DeleteasmuchofthepolylinkeraspossIBLeassometimestransposon"hot-spots"intoit.
- SelecttransformantsonLBKan40.
TransformthisplasmidintocompetentcellsofR1236/B211.
- SelectonLBKan40Cm34.
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.
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-70stockofbacterialpoolforfutureuse).
- TransformNotIdigestofentirepoolintoyeast,selectingforURA3..LookforlacZfusionsamongtransformants.
- ForHATepitope-tagging,youmaywanttopre-transformyouryeaststrainwithpB227/GAL-cre(selectingLEU2).

Screeningforin-framelacZfusionsinyeast

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

TransformstrainwithplasmidpB227/GAL-cre,selectingonSC-leu.
Inoculatetransformantsinto2mlsSC-ura-leuwith2%raffinoseascarbonsource,andgrowtosaturation.
Dilute1/100intoSC-leuwith2%galactoseascarbonsource.Asacontrolalsodilute1/100intoSC-leuwith2%glucoseascarbonsource.Growfor2days(somestrainsinducewithoutgrowing).
Ifgrown,dilute1/100.Otherwise,proceedwithundilutedculture.
- Spota10uldropontoanFOAplateandstreakitforsinglecolonies(non-quantitativeapproach!).
- Alternatively,platedilutionsontoSCmediaandreplicatoSC-uratoidentifyUra-colonies.Theinducedculturesshouldgive100-foldmoreUra-cellsthanthecontrol.
PCRprimersdesignedusingthesequencegivenbelowcanbeusedtodeterminepositionofthetag.TheTRelementsandpalindromicloxRregionshouldbeavoided.

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

SequenceofHATtag(3xHA):

38bpTerminalRepeatinuppercase.loxRinbold.

GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGgcggccattgaaggtagaagagaaAATttgtacttccaaagaaagaaggccgctatcgcttcggataactcctgctatacgaagttatgggcggccgtttacccatacgatgttcctgactatgcgggctatccctatgacgtcccggactatgcaggATCCtatccatatgacgttccagattacgctccggccgccCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC

Bacterialstrainsused(providedinkit):

R1123	StrainXL1-bluecarryingvectorpHSS6.
R1236/B211	StrainRDP146(F^-recA"(deltalac-pro)rpsE;spectinomycinresistant)withplasmidpLB101(pACYC184withtnpA;activetransposase,chloramphenicolresistant)(F.Heffron)
#95/B427	StrainRDP146withpOX38FfactorderivativecarryingmTn3derivativemTn-3xHA/lacZ(lacZ,URA3,tet;tetracyclineresistant)
#70/B425	StrainNG135(K12recA56gal-delS165strA;streptomycinresistant)withplasmidpNG54(pACYC184withmTn3resandtnpRseqs;activeresolvase,chloramphenicolresistant)(N.Grindley)
B227	StrainDH5-alphacarryingpB227/GAL-cre(amp,ori,CEN,LEU2)(B.Sauer)

Vectorused:

TheaccessionforpHSS6isM84115.

Antibioticsused:

Tetracycline,Tet(SigmaT3383)	12mg/mlinwater.Useat3ug/ml(Tet3)
Kanamycin,Kan(SigmaK800)	10mg/mlinwater.Useat40ug/ml(Kan40)
Chloramphenicol,Cm(SigmaC0378,Ithink)	34mg/mlinethanol.Useat34ug/ml(Cm34)
Streptomycin,Sm(SigmaS6501)	10mg/mlinwater.Useat50ug/ml(Sm50)
Ampicillin,Amp(SigmaA9518)	50mg/mlinwater.Useat50ug/ml(Amp50)

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

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