Theoriginallibrarywasdistributedas18individualpools.After,thereisanewlibrarythatisdistributedas10pools.YouwillbesentaboutamicrogramofeachpoolintheformofDNA.TransformasuitableamountintoE.coli(useanykanamycin-andtetracycline-sensitivestrainsuitableformakingplasmidpreps).Selecttransformantswith40ug/mlkanamycinand/or3ug/mltetracycline(allowatleastanhourforexpressionfollowingtransformation).Obtain50,000coloniesforeachpool.ElutecoloniesfromplatesinLB;makea-70oCstockofthiseluate.DiluteeluateintoLBplusantibiotictogiveaculturewithanalmostsaturateddensity.Growat37oCforafewhours.MakeminiprepormidiprepDNA. OVERVIEW:MutagenizedDNAfromthelibraryisexcisedfromthebacterialvector.Itisthentransformedintoaura3strainofyeast.Thisprocedureisoutlinedinthisfigure.Thebeststrategyistoscreenafewthousandtransformantsfromeachpool.Useofacircle-zerostrainwillpreventrecoveryofinsertionsinthe2-micronplasmid.Forlibrariesdistributedafter,thegenomiclibrarywasmadefromacircle-zerostrain(alsorho-minus)sothisisnotnecessary.Screening30,000transformantsshouldgiveyou95%coverageoftheyeastgenome. Tominimizedoubleintegrants,transformationsshouldcontainthelowestamountofDNApracticable.Wethereforerecommendthatapilotexperimentbeperformedtodeterminetransformationefficiencyofthestrain,andconditionsthenbescaledupasappropriate.ThepilotprotocolgivenbelowusesamodifiedversionofthemethodofChenetal.(1992).Youshouldusewhatevertransformationprotocolworksbestinyourhands. OVERVIEW:Transformantstrainscarryingin-framefusionsbetweenyeastgenesandlacZareidentifiedbyacolorassayforbeta-galactosidaseactivity. OVERVIEW:Todeterminethesiteoftransposoninsertion,genomicDNAimediatelyadjacenttothelacZsequencesisrescuedinEscherischiacoli(seefigure).Tointroduceanoriginofreplication(ori),aplasmidmarkedwithLEU2(pRSQ2-LEU2,U64693)replacespartofthetransposonbyrecombinationbetweenplasmid-andtransposon-bornecopiesoflacZsequences.YeastDNAisrecoveredfromthesetransformantsandcutwitha"recovery"enzyme(EcoRI,HindIII,ClaI,SalI,XhoI,KpnI).Thisreleasesasalinearsegmentthebacterialoriginofreplication,thebeta-lactamasegeneandaportionofthelacZgenewithadjacentyeastDNA;thisfragmentisthencircularizedandrecoveredinbacteria.pRSQ2ishighcopynumberinE.coli.Plasmidsaresequencedusingaprimercomplementarytothe5"endofthetransposon.Thisprocessrequiresthethreeprotocolsgivenbelow. Alternativemethod:CarlFriddle(http://genome-www.stanford.edu/group/botlab)hasdevelopedavectorettePCRrescueprotocolforlacZ-basedtransposons.IhavetranscribedCarl"sprotocolandmodifiedthesuggestedenzymesandprimers,tomakeitsuitableforvectorettePCRofthemTn-3xHA-basedtransposons. CAUTION!Twoormoreinsertioneventsmayhaveoccurred.Thesecanbeidentifiedbyexaminationofsegregationofthetransposon-borneURA3Markerupontetraddissection.YoushouldbesurethatthestrainhasonlyonetransposonbeforeproceedingtorecoveringaplasmidcontaininggenomicDNA.Ifyouhaveusedthelibraryformutagenesis,youarestronglyadvisedtomakesurethatyourphenotypeislinkedtothetransposoninsertion,sincespontaneousmutationscanariseatothersites.Youcanwastetimerecoveringjunkifyoudon"tcheck. Wesuggesttransformingtheyeastwith1-5ugofBamHI-digestedpRSQ2-LEUplasmidDNA,selectingthetransformantsonSC-leu-ura.Thisisatargettedreplacement,soefficiencywilldependonyourstrain.ThemethodofChenetal.(1992)givenabovecanbeused.IfanampRplasmidispresentintheyeaststraintobetransformed,adifferentmarkercouldbeclonedintothepRSQ2polylinkertoenableitsrecovery. ForadetaileddiscussionofgenomicDNApreparationfromyeast,seePhilippsenetal.(1991).Hereisthemethodthatweuse. Uppercaseindicatesterminalrepeatelementalsopresentinvector: GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGgcggccattgaaggtagaag TheaccessionformTn-3xHA/lacZisU54828. WhenpRSQ2-LEU2integratesintomTn-3xHAitcreatesan11.8kbinsertion.Thiselementisnotcleavedbythefollowingenzymes:AscI,AvrII,BspEI,MscI,NotI,PmeI,PmlI,SacII,SnABI,SpeI.Theseenzymescanthereforebeusedtorecoveralargeplasmidcontainingsequencesboth5"and3"tothetransposoninsertion.Wehavesuccessfully"moved"disruptionsbythisstrategy. Whentransposoninsertionhascreatedanin-framefusiontolacZinthegeneofinterest,thetransposoncanbeexcizedtoleavea274bpinsertion(sequencegivenbelow)containingthe3xHAtag.Withthe5basepairduplicationcausedbytransposoninsertion,thisgivesanin-frame93-aminoacidinsertionintheprotein.ThepopouteventismediatedbycrerecombinaseandrequiresinductionoftheGAL1-10promoterongalactose.Ourstrainsgrowpoorlyongalactosebutgive80to100%popouts. TheHAtripletagcanbedetectedbymousemonoclonalantibodies12CA5(Boehringer)orMMS101R(BAbCo,Richmond,California).Theseantibodyrecognisecross-reactingyeastproteinsofabout55kDor110kD,respectively,andcangiveaspottybackgroundonimmunofluorescence.Despitethisdrawback,the3xHAtaghasbeenusedextensivelyandsuccessfullyinyeast.Arabbitpolyclonalantiseraisalsoavailable(101c500;BabCo)butthiswaslessreactiveintheoneinstancewetried.Protocolsforyeastimmunofluorescencecanbefoundhere,orinMethodsinEnzymology194(1991). N.B.Whentaggingessentialgenes,theoriginalstraintransformedshouldobviouslybediploid.Youcandissectthepopped-outversiontoseeifthetaggedgeneisfunctional.Onlybelieveatagislethalifitiscomplementedbythewild-typegene,andifseveralpopouteventsgivethesamephenotype. TRinuppercase.loxRinbold. GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGgcggccattgaaggtagaagagaaaatttgtacttccaaagaaagaaggccgctatcgcttcggataactcctgctatacgaagttatgggcggccgtttacccatacgatgttcctgactatgcgggctatccctatgacgtcccggactatgcaggATCCtatccatatgacgttccagattacgctccggccgcCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCMakinglibraryDNAfromtheDNAwesendyou
TransformingyeastwithDNAfromtheinsertionlibrary
ScreeningforgeneexpressionusinglacZfusions
Identificationofthegenomicsiteoftransposoninsertion
Transformationofyeaststrains
RecoveryofgenomicDNAfromyeaststrains
Plasmidrescue
SequenceoflacZendofmTn3-3xHA
Transferringthedisruptionalleletootherstrains:
UsingtheHATepitopetaggingfeatureofmTn-3xHA/lacZ
SequenceofHATtag(3xHA):
GenBankaccession
Antibioticsused:
Tetracycline,Tet(SigmaT3383) 12mg/mlin50%ethanol.Useat3ug/ml(Tet3) Kanamycin,Kan(SigmaK800) 10mg/mlinwater.Useat40ug/ml(Kan40) Ampicillin,Amp(SigmaA9518) 50mg/mlinwater.Useat50ug/ml(Amp50)