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mTn4xHA/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. Pleasereadthiswholedocumentbeforeyoustart! 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). N.B.Whentaggingessentialgenes,theoriginalstraintransformedshouldobviouslybediploid.YoucandissecttheHAT-taggedversiontoseeifthetaggedgeneisfunctional.Toberigourous,onlybelieveatagislethalifitiscomplementedbythewild-typegene,andifseveralpopouteventsgivethesamephenotype. TRinuppercase.loxRinbold. GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGgcggcctacccatacgacgtcccagactacgcgttggccgctatcgcttcggataactcctgctatacgaagttatgggcggccgtttacccatacgatgttcctgactatgcgggctATCCctatgacgtcccggactatgcaggatcctatccatatgacgttccagattacgctccggccgcCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC TheaccessionforpHSS6isM84115. Whenonlyafewplatesofeachtypeareused,it"sconvenienttochopanLBplateupwithasteriletoothpick,putthebitsinasterileflask,andmelttheagarbymicrowave.Addappropriateamountsofantibioticandrepourplates.mTn-4xHA/lacZ
TR Tn3terminalinvertedrepeats HA Hemagglutinin(HA)epitope loxR loxsite,targetforCrerecombinase lacZ 5"-truncatedlacZgeneencodingbeta-galactosidase URA3 URA3genefromS.cerevisiae tet Tetracyclineresistancegene res Tn3siteforresolutionoftranspositionintermediate loxP loxsite,targetforCrerecombinase 3xHA Hemagglutinin(HA)tripleepitopetag Shuttlemutagenesis
Screeningforin-framelacZfusionsinyeast
UsingtheexcisionfeaturetoHAT-epitopetagaprotein
SequenceofHATtag(4xHA):
Bacterialstrainsused(providedinkit):
R1123 StrainXL1-bluecarryingvectorpHSS6 R1236/B211 StrainRDP146(F-recA"(deletionlac-pro)rpsE;spectinomycinresistant)withplasmidpLB101(pACYC184withtnpA;activetransposase,chloramphenicolresistant)(F.Heffron) #94/B426 StrainRDP146withpOX38FfactorderivativecarryingmTn3derivativemTn-4xHA/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:
Antibioticsused:
Tetracycline,Tet(SigmaT3383) 12mg/mlin50%ethanol.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)
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