大肠杆菌不同菌株的基因型解读

大肠杆菌不同菌株的基因型解读E.coligenotypesContents[hide]1Nomenclature&Abbreviations2MethylationIssuesinE.coli3Commonlyusedstrains3.1AG13.2AB11573.3BL213.4BL21(AI)3.5BL21(DE3)3.6BL21(DE3)pLysS3.7BNN933.8BNN973.9BW26434,CGSCStrain#76583.10C6003.11C600hflA150(Y1073,BNN102)3.12CSH503.13D12103.14DB3.13.15DH13.16DH5α3.17DH10B(Invitrogen)3.18DH12S(Invitrogen)3.19DM1(Invitrogen)3.20E.cloni(r)5alpha(Lucigen)3.21E.cloni(r)10G(Lucigen)3.22E.cloni(r)10GF'(Lucigen)3.23E.coliK12ER2738(NEB)3.24ER2566(NEB)3.25ER2267(NEB)3.26HB1013.27HMS174(DE3)3.28High-Control(tm)BL21(DE3)(Lucigen)3.29High-Control(tm)10G(Lucigen)3.30IJ11263.31IJ11273.32JM833.33JM1013.34JM1033.35JM1053.36JM1063.37JM1073.38JM1083.39JM1093.40JM109(DE3)3.41JM1103.42JM2.3003.43LE3923.44Mach13.45MC10613.46MC41003.47MG16553.48OmniMAX23.49OverExpress(tm)C41(DE3)(Lucigen)3.50OverExpress(tm)C41(DE3)pLysS(Lucigen)3.51OverExpress(tm)C43(DE3)(Lucigen)3.52OverExpress(tm)C43(DE3)pLysS(Lucigen)3.53Rosetta(DE3)pLysS3.54Rosetta-gami(DE3)pLysS3.55RR13.56RV3083.57SOLR(Stratagene)3.58SS320(Lucigen)3.59STBL2(Invitrogen)3.60STBL3(Invitrogen)3.61STBL43.62SURE(Stratagene)3.63SURE2(Stratagene)3.64TG1(Lucigen)3.65TOP10(Invitrogen)3.66Top10F'(Invitrogen)3.67W31103.68XL1-Blue(Stratagene)3.69XL1-BlueMRF'(Stratagene)3.70XL2-Blue(Stratagene)3.71XL2-BlueMRF'(Stratagene)3.72XL1-Red(Stratagene)3.73XL10-Gold(Stratagene)3.74XL10-GoldKanR(Stratagene)4Othergenotypeinformationsources5ReferencesNomenclature&AbbreviationsAlistedgenenamemeansthatgenecarriesalossoffunctionmutation,aΔprecedingagenenamemeansthegeneisdeleted.Ifageneisnotlisted,itisnotknowntobemutated.ProphagespresentinwtK-12strains(F,λ,e14,rac)arelistedonlyifabsent.E.coliBstrainsarenaturallylon-anddcm-.F-=DoesnotcarrytheFplasmidF+=CarriestheFplasmid.ThecellisabletomatewithF-throughconjugation.F'[]=CarriesanFplasmidthathashostchromosomalgenesonitfromapreviousrecombinationevent.ThiscellcanalsomatewithF-throughconjugation.ChromosomalgenescarriedintheFplasmidarelistedinbrackets.rB/K+/-=The(B/K)definesthestrainlineage.The+/-indicateswhetherthestrainhasorhasn'tgottherestrictionsystem.mB/K+/-=The(B/K)definesthestrainlineage.The+/-indicateswhetherthestrainhasorhasn'tgotthemodification(methylation)system.hsdS=Bothrestrictionandmethylationofcertainsequencesisdeletedfromthestrain.IfyoutransformDNAfromsuchastrainintoawildtypestrain,itwillbedegraded.hsdR=ForefficienttransformationofclonedunmethylatedDNAfromPCRamplificationsINV()=chromosomalinversionbetweenlocationsindicatedahpC=mutationtoalkylhydroperoxidereductaseconferringdisulfidereductaseactivityara-14=cannotmetabolizearabinosearaD=mutationinL-ribulose-phosphate4-epimeraseblocksarabinosemetabolismcycA=mutationinalaninetransporter;cannotusealanineasacarbonsourcedapD=mutationinsuccinyldiaminopimelateaminotransferaseleadstosuccinateor(lysine+methionine)requirementΔ()=chromosomaldeletionofgenesbetweenthelistedgenes(mayincludeunlistedgenes!)dam=adeninemethylationatGATCsequencesexist;highrecombinationefficiency;DNArepairturnedondcm=cytosinemethylationatsecondCofCCWGGsitesexist.dam&dcmarethedefaultpropertiesandalwayselided,whiledam-ordcm-shouldbedeclareexplicitlydeoR=regulatorygenethatallowsconstitutiveexpressionofdeoxyribosesynthesisgenes;permitsuptakeoflargeplasmids.SeeHanahanD,USPatent4,851,348.***Thishasbeencalledintoquestion,astheDH10BgenomesequencerevealedthatitisdeoR+.SeeDurfee08,PMID18245285.dnaJ=oneofthechaparoninsinactivated;stabilizessomemutantproteinsdut1=dUTPaseactivityabolished,leadingtoincreaseddUTPconcentrations,allowinguracilinsteadofthymineincorporationinDNA.StableUincorporationrequiresunggenemutationaswell.endA1=ForcleanerpreparationsofDNAandbetterresultsindownstreamapplicationsduetotheeliminationofnon-specificdigestionbyEndonucleaseI(e14)=excisableprophagelikeelementcontainingmcrAgene;presentinK-12butmissinginmanyotherstrainsgalE=mutationsareassociatedwithhighcompetence,increasedresistancetophageP1infection,and2-deoxygalactoseresistance.galEmutationsblocktheproductionofUDP-galactose,resultingintruncationofLPSglycanstotheminimal,"innercore".TheexceptionalcompetenceofDH10B/TOP10isthoughttobearesultofareducedinterferencefromLPSinthebindingand/oruptakeoftransformingDNA.galE15isapointmutationresultinginaSer123->Pheconversionneartheenzyme'sactivesite.SeevanDie,etal.PMID6373734,Hanahan,etal.PMID1943786,andEcoSalISBN1555811647.--Dcekiert16:56,23January2008(CST)galk=mutantscannotmetabolizegalactoseandareresistantto2-deoxygalactose.galK16isanIS2insertion~170bpdownstreamofthegalKstartcodon.SeeEcoSalISBN1555811647.--Dcekiert16:56,23January2008(CST)galU=mutantscannotmetabolizegalactosegor=mutationinglutathionereductase;enhancesdisulphidebondformationglnV=suppressionofamber(UAG)stopcodonsbyinsertionofglutamine;requiredforsomephagegrowthgyrA96=mutationinDNAgyrase;conveysnalidixicacidresistancegyrA462=mutationinDNAgyrase;conveysresistancetoccdBcolicingeneproducthflA150=proteasemutationstabilizingphagecIIprotein;highfrequencyoflysogenizationbyλΔ(lac)X74=DeletionoftheentirelacoperonaswellassomeflankingDNA(completedeletionisΔcod-mhpF;seeMol.Micro.,6:1335,andJ.Bact.,179:2573)lacIqorlacIQ=overproductionofthelacrepressorprotein;-35siteinpromoterupstreamoflacIismutatedfromGCGCAAtoGTGCAAlacIQ1=overproductionofthelacrepressorprotein;containsa15bpdeletiontocreateoptimal-35siteinpromoterupstreamoflacIlacY=deficientinlactosetransport;deletionoflactosepermease(Mprotein)lacZΔM15=partialdeletionofthelacZgenethatallowsαcomplementationoftheβ-galactosidasegene;requiredforblue/whiteselectiononXGalplates.DeletestheaminoportionoflacZ(aa11-41).leuB=requiresleucineΔlon=deletionofthelonproteasemalA=cannotmetabolizemaltosemcrA=MutationeliminatingrestrictionofDNAmethylatedatthesequenceCmCGG(possiblymCG).Carriedonthee14prophage(q.v.)mcrB=MutationeliminatingrestrictionofDNAmethylatedatthesequenceRmCmetB=requiresmethioninemetC=requiresmethioninemrr=MutationeliminatingrestrictionofDNAmethylatedatthesequenceCmAGorGmACmtlA=cannotmetabilizemannitol(Mu)=Muprophagepresent.Muδmeansthephageisdefective.mutS-mutationinhibitsDNArepairofmismatchesinunmethylatednewlysynthesizedstrandsnupG=sameasdeoRompT=mutationinoutermembraneproteinproteaseVII,reducingproteolysisofexpressedproteins(P1)=CellcarriesaP1prophage.CellsexpresstheP1restrictionsystem.(P2)=CellcarriesaP2prophage.AllowsselectionagainstRed+Gam+λ(φ80)=Cellcarriesthelambdoidprophageφ80.AdefectiveversionofthisphagecarryinglacZM15deletion(aswellaswild-typelacI,lacYA,andflankingsequences)ispresentinsomestrains.Theφ80attachmentsiteisjustadjacenttotonB.pLysS=containspLysSplasmidcarryingchloramphenicolresistanceandphageT7lysozyme,effectiveatattenuatingactivityofT7RNApolymerase,forbetterinhibitionofexpressionundernon-inducedconditions.Thesequencecanbefoundhere.proA/B=requiresprolinerecA1=ForreducedoccurrenceofunwantedrecombinationinclonedDNA;cellsUVsensitive,deficientinDNArepairrecA13=asforrecA1,butinsertslessstable.recBCD=ExonucleaseV;mutationinRecBorRecCreducesgeneralrecombinationbyafactorof100;impairedDNArepair;UVsensitive,easierpropagationofinvertedrepeatsrecJExonucleaseinvolvedinalternaterecombinationrelA=relaxedphenotype;permitsRNAsynthesisinabsenceofproteinsynthesisrha=blockedrhamosemetabolismrnc=encodesRnaseIII(rnc-14isacommonnullmutant)rne=encodesRnaseE(rne-3071isacommontemperaturesensitivemutant)rpsL=mutationinribosomalproteinS12conveyingstreptomycinresistance;alsocalledstrAsbcBC=ExoIactivityabolished;usuallypresentinrecBCstrains;recombinationproficient,stableinvertedrepeatssr1=cannotmetabolizesorbitolsupE=glnVsupF=tyrTthi=requiresthiaminethyA=requiresthymidineTn10=transposonnormallycarryingTetracyclineresistanceTn5=transposonnormallycarryingKanamycinresistancetonA=MutationinoutermembraneproteinconveyingresistancetophageT1andphageT5traD=Mutationeliminatingtransferfactor;preventstransferofFplasmidtrxB=mutationinthioredoxinreductase;enhancesdisulphidebondformationinthecytoplasmtsx=outermembraneproteinmutationconveyingresistancetophageT6andcolicinKtyrT=suppressionofamber(UAG)stopcodonsbyinsertionoftyrosine;neededforsomephageinfectionsuchasλgt11.ung1=allowsuraciltoexistinplasmidDNAxyl-5=blockedxylosemetabolismSmR=StreptomycinresistanceMethylationIssuesinE.coliTypeImethylationsystems:E.coliK-12restrictsDNAwhichisnotprotectedbyadeninemethylationatsitesAA*C[N6]GTGCorGCA*C[N6]GTT,encodedbythehsdRMSgenes(EcoKI).Deletionsinthesegenesremoveseithertherestrictionormethylationorbothofthesefunctions.E.coliBderivativestrainscontainanhsdRMSsystem(EcoBI)restrictingandprotectiingthesequenceTGA*[N8]TGCTorAGCA*[N8]TCA.ThemcrAgene(carriedonthee14prophage)restrictsDNAwhichismethylatedinCmCWGGormCGsequences(methylationbythedcmgeneproduct).ThemcrBCgenesrestrictRmCsequences.ThemrrgeneproductrestrictsadeninemethylatedsequencesatCAGorGACsites.E.colimethylatestheadenineinGATC(andthecorrespondingAontheoppositestrand)withthedamgeneproduct.M.EcoKIImethylatesthefirstAatthepalindromicsiteATGCAT(aswellasthecorrespondingAontheoppositestrand),see(KossykhVG(2004)J.Bact186:2061-2067PMID15028690)Notethatthisarticlehasbeenretracted;theretractionappearstocenterontextualplagarism,notexperimentalresults.ThehomologytoAvaIIIisreal.IthinkIbelieveit.tk20:28,9December2005(EST).RichRobertsreports:"Wehavetriedourselvestodetectactivitywiththisgeneproductandcannotdetectanymethyltransferaseactivity.InourcaseweusedantibodiesabletodetectN6-methyladenineorN4methylcytosineinDNA.Theoneswehaveareverysensitiveandshouldhavebeenabletodetect5methylgroupsinthewholeE.colichromosome.Nothingwasdetectedinanoverexpressingstrain."ForadditionalinformationseeE.colirestriction-modificationsystemandtheNEBtechnicalinformationonmethylation.CommonlyusedstrainsAG1endA1recA1gyrA96thi-1relA1glnV44hsdR17(rK-mK+)AB1157thr-1,araC14,leuB6(Am),Δ(gpt-proA)62,lacY1,tsx-33,qsr'-0,glnV44(AS),galK2(Oc),LAM-,Rac-0,hisG4(Oc),rfbC1,mgl-51,rpoS396(Am),rpsL31(strR),kdgK51,xylA5,mtl-1,argE3(Oc),thi-1BachmannBJ:DerivationandgenotypesofsomemutantderivativesofEscherichiacoliK-12.TheHI-Control10Gcellscontainasingle-copyBACplasmidharboringaspeciallyengineeredversionofthelacIq1repressorallele.ThelacIq1alleleexpresses~170-foldmorelacrepressorproteinthanthewild-typelacIgene.ForstablecloningofT7proteinexpressionplasmids.ResistanttophageT1.IJ1126E.coliK-12recB21recC22sbcA5endAgalthiSu+Δ(mcrC-mrr)102::Tn10SeeEndy:IJ1126IJ1127IJ1126lacUV5lacZ::T7gene1-KnrSeeEndy:IJ1127JM83rpsLaraΔ(lac-proAB)Φ80dlacZΔM15Sigmaliststhi.Checkthis.streptomycinresistantJM101glnV44thi-1Δ(lac-proAB)F'[lacIqZΔM15traD36proAB+]hostforM13mpvectorsrecA+,rK+originalblue/whitecloningstrainhasallwtrestrictionsystemsReferences:Messing,J.etal.(1981)NucleicAcidsRes.9,309;Yanisch-Perron,C.,Vieira,J.,andMessing,J.(1985)Gene33,103.JM103endA1glnV44sbcBCrpsLthi-1Δ(lac-proAB)F'[traD36proAB+lacIqlacZΔM15]streptomycinresistantReferences:Hanahan,D.(1983)J.Mol.Biol.166:557-80.NEBsaysthisstrainencodesaprophageencodedEcoP1endonuclease.Sigmalists(P1)(rK-mK+rP1+mP1+)JM105endA1glnV44sbcB15rpsLthi-1Δ(lac-proAB)[F'traD36proAB+lacIqlacZΔM15]hsdR4(rK-mK+)SigmalistssbcCstreptomycinresistantReferences:Yanisch-Perron,C.,Vieira,J.,andMessing,J.(1985)Gene33,103.JM106endA1glnV44thi-1relA1gyrA96Δ(lac-proAB)F-hsdR17(rK-mK+)References:Yanisch-Perron,C.,Vieira,J.,andMessing,J.(1985)Gene33,103.JM107endA1glnV44thi-1relA1gyrA96Δ(lac-proAB)[F'traD36proAB+lacIqlacZΔM15]hsdR17(RK-mK+)λ-NEBGeneticStockCenter:OneofthereasonstheJM109+λ(DE3)DE3prophagecarryingT7polymeraseexpressioncassetteSamecassetteasBL21(DE3)carryingalacinducibleT7RNApolymeraseandlacIqnalidixicacidresistantJM110rpsLthrleuthilacYgalKgalTaratonAtsxdamdcmglnV44Δ(lac-proAB)e14-[F'traD36proAB+lacIqlacZΔM15]hsdR17(rK-mK+)SigmafailstolisttonAtsxe14fhuAhsdR17(e14-)statusuncertainstreptomycinresistantJM2.300lacI22,LAM-,e14-,rpsL135(strR),malT1(LamR),xyl-7,mtl-1,thi-1Somefolkshavebeenusingthisstrain(i.e.,Elowitz,Gardner)andittookmetoolongtofindtheCGSC#.ThisstrainisnolongeravailablefromtheCGSCLE392glnV44supF58(lacY1orΔlacZY)galK2galT22metB1trpR55hsdR514(rK-mK+)SigmalistsF-e14-Mach1ΔrecA1398endA1tonAΦ80ΔlacM15ΔlacX74hsdR(rK-mK+)FromInvitrogenDoublingtimeapprox.50minandsupposedlyfastestgrowingchemicallycompetentcloningstrainavailableMach1cellsarederivativesofE.coliWstrains(ATCC9637,S.A.Waksman),ratherthanE.coliK-12.ThismayhaveimplicationsforBL-1statusforsomefacilities(apparentlynotforMIT).SeeBloom04patentfordetailsontheconstructionandpropertiesofthisstrain.MC1061F-Δ(ara-leu)7697[araD139]B/rΔ(codB-lacI)3galK16galE15λ-e14-mcrA0relA1rpsL150(strR)spoT1mcrB1hsdR2(r-m+)StreptomycinresistantThethr-leuregionwastransducedfromanE.coliB/rstrain(SB3118)inearlystepsofstrainconstruction.ParentofDH10B/TOP10andderivedstrainsReferences:E.coliGeneticStockCenter,MC1061RecordCaCGSC#1157productpageformoreinformation.Wood,1966)bytransducingtoMetpaperorthesummaryinMethodsinEnzymologyformoredetails.[1]BW26434,CGSCStrain#7658Δ(araD-araB)567,Δ(lacA-lacZ)514(::kan),lacIp-4000(lacIq),λ-,rpoS396(Am)?,rph-1,Δ(rhaD-rhaB)568,hsdR514ThisinformationisfromaprintoutsentbytheE.coliGeneticStockCenterwiththestrain.B.L.Wannerstrainrph-1isa1bpdeletionthatresultsinaframeshiftoverlast15codonsandhasapolareffectonpyrEleadingtosuboptimalpyrimidinelevelsonminimalmedium.(Jensen1993JBact.175:3401)Δ(araD-araB)567wasformerlycalledΔaraBADAH33byDatsenkoandWannerAm=amber(UAG)mutationReference:DatsenkoandWanner,2000,PNAS,97:6640NOTE:ThispromoterdrivingtheexpressionoflacIwassequencedinthisstrainusingaprimerinmhpR(upstreamoflacI)andaprimerintheoppositeorientationinlacI.Thelacpromoterwasfoundtobeidenticaltowildtype.Thus,the-35sequencewasGCGCAAnotGTGCAAasexpectedwithlacIq.Thereforethisstrain(oratleasttheversionobtainedfromtheE.coliGeneticStockCenter)doesNOTappeartobelacIq.AccordingtoBarryWanner,thisisanunexpectedresult.-Reshma13:19,5May2005(EDT)"WehavenowconfirmedthatBW25113,BW25141,andBW26434arealllacI+,andnotlacIq.WethankyouforalertingustotheerrorwithrespecttoBW26434.Apparently,thelacIregionwasrestoredtowild-typeinapredecessorofBW25113."(fromBarryWannerNovember18,2005)ThegenotypehasbeencorrectedattheCGSCC600F-tonA21thi-1thr-1leuB6lacY1glnV44rfbC1fhuA1λ-Therearestrainscirculatingwithbothe14+(mcrA+)ande14-(mcrA-)GeneralpurposehostSeeCGSC#3004References:Appleyard,R.K.(1954)Genetics39,440;Hanahan,D.(1983)J.Mol.Biol.166,577.C600hflA150(Y1073,BNN102)F-thi-1thr-1leuB6lacY1tonA21glnV44λ-hflA150(chr::Tn10)hostforrepressingplaquesofλgt10whenestablishingcDNAlibrariesReferenceYoungR.A.andDavis,R.(1983)Proc.Natl.Acad.Sci.USA80,1194.TetracyclineresistancefromtheTn10insertionCSH50F-λ-araΔ(lac-pro)rpsLthifimE::IS1SeeCGSC#8085References:Miller,J.H.1972.Expts.inMolec.Genetics,CSH0:14-0;Blomfeldetal.,J.Bact.173:5298-5307,1991.D1210HB101lacIqlacY+DB3.1F-gyrA462endA1glnV44Δ(sr1-recA)mcrBmrrhsdS20(rB-,mB-)ara14galK2lacY1proA2rpsL20(Smr)xyl5Δleumtl1usefulforpropagatingplasmidscontainingtheccdBoperon.gyrA462enablesccdBcontainingplasmidpropagationstreptomycinresistantappearstoNOTcontainlacI(basedonacolonyPCR)--AustinChe16:16,18June2007(EDT)1.Bernard-JMolBiol-1992pmid=13243242.Miki-JMolBiol-1992pmid=1316444DH1endA1recA1gyrA96thi-1glnV44relA1hsdR17(rK-mK+)λ-parentofDH5αAnHoffman-Berling1100strainderivative(Meselson68)moreefficientattransforminglarge(40-60Kb)plasmidsnalidixicacidresistantReference:MeselsonM.andYuanR.(1968)Nature217:1110PMID4868368.DH5αF-endA1glnV44thi-1recA1relA1gyrA96deoRnupGΦ80dlacZΔM15Δ(lacZYA-argF)U169,hsdR17(rK-mK+),λ–AnHoffman-Berling1100strainderivative(Meselson68)PromegaalsolistsphoAnalidixicacidresistantReferences:FOCUS(1986)8:2,9.Hanahan,D.(1985)inDNACloning:APracticalApproach(Glover,D.M.,ed.),Vol.1,p.109,IRLPress,McLean,Virginia.Grant,S.G.N.etal.(1990)Proc.Natl.Acad.Sci.USA87:4645-4649PMID2162051.MeselsonM.andYuanR.(1968)Nature217:1110PMID4868368.DH10B(Invitrogen)F-endA1recA1galE15galK16nupGrpsLΔlacX74Φ80lacZΔM15araD139Δ(ara,leu)7697mcrAΔ(mrr-hsdRMS-mcrBC)λ-suitableforcloningmethylatedcytosineoradeninecontainingDNAanMC1061derivative(Casadaban80).PreparecellsforchemicaltransformationwithCCMB80bufferblue/whiteselectionWhileDH10BhasbeenclassicallyreportedtobegalUgalK,thepreliminarygenomesequenceforDH10BindicatesthatDH10B(andbytheirlineagealsoTOP10andanyotherMC1061derivatives)isactuallygalEgalKgalU+.Dcekiert16:37,23January2008(CST)GenomesequenceindicatesthatDH10BisactuallydeoR+.PresumablyTOP10andMC1061arealsodeoR+.StreptomycinresistantReferences:Casdaban,M.andCohen,S.(1980)JMolBiol138:179PMID6997493.Grant,S.G.N.etal.(1990)Proc.Natl.Acad.Sci.USA87:4645-4649PMID2162051.E.coliGeneticStockCenter,MC1061RecordDH10BGenomeSequencingProject,BaylorCollegeofMedicineCompletesequenceisavailable,seeDurfee08,PMID18245285.DH12S(Invitrogen)mcrAΔ(mrr-hsdRMS-mcrBC)φ80dlacZΔM15ΔlacX74recA1deoRΔ(ara,leu)7697araD139galUgalKrpsLF'[proAB+lacIqZΔM15]hostforphagemidandM13vectorsusefulforgeneratinggenomiclibrariescontainingmethylatedcytosineoradenineresiduesstreptomycinresistantReferences:Lin,J.J.,Smith,M.,Jessee,J.,andBloom,F.(1991)FOCUS13,96.;Lin,J.J.,Smith,M.,Jessee,J.,andBloom,F.(1992)BioTechniques12,718.DM1(Invitrogen)F-dam-13::Tn9(CmR)dcm-mcrBhsdR-M+gal1gal2ara-lac-thr-leu-tonRtsxRSu0HostforpBR322andothernon-pUC19plasmids;usefulforgeneratingplasmidsthatcanbecleavedwithdamanddcmsensitiveenzymesChloramphenicolresistantPromegalistsasF'notF-Reference:Lorow-MurrayDandBloomF(1991)Focus13:20E.cloni(r)5alpha(Lucigen)fhuA2Δ(argF-lacZ)U169phoAglnV44Φ80Δ(lacZ)M15gyrA96recA1relA1endA1thi-1hsdR17Commoncloningstrain.E.cloni(r)10G(Lucigen)F-mcrAΔ(mrr-hsdRMS-mcrBC)endA1recA1Φ80dlacZΔM15ΔlacX74araD139Δ(ara,leu)7697galUgalKrpsLnupGλ-tonACommoncloningstrain.ResistanttophageT1.E.cloni(r)10GF'(Lucigen)[F´proA+B+lacIqZΔM15::Tn10(TetR)]/mcrAΔ(mrr-hsdRMS-mcrBC)endA1recA1Φ80dlacZΔM15ΔlacX74araD139Δ(ara,leu)7697galUgalKrpsLnupGλtonAStrainforcloningandsingle-strandDNAproduction.E.coliK12ER2738(NEB)F´proA+B+lacIqΔ(lacZ)M15zzf::Tn10(TetR)/fhuA2glnVΔ(lac-proAB)thi-1Δ(hsdS-mcrB)5PhagepropagationstrainAlsoavailablefromLucigenCorporation.ER2566(NEB)F-λ-fhuA2[lon]ompTlacZ::T7gene1galsulA11Δ(mcrC-mrr)114::IS10R(mcr-73::miniTn10-TetS)2R(zgb-210::Tn10)(TetS)endA1[dcm]HoststrainfortheexpressionofatargetgeneclonedinthepTYBvectors.CarryachromosomalcopyoftheT7RNApolymerasegeneinsertedintolacZgeneandthusunderthecontrolofthelacpromoter.IntheabsenceofIPTGinductionexpressionofT7RNApolymeraseissuppressedbythebindingoflacIrepressortothelacpromoter.DeficientinbothlonandompTproteases.ER2267(NEB)F´proA+B+lacIqΔ(lacZ)M15zzf::mini-Tn10(KanR)/Δ(argF-lacZ)U169glnV44e14-(McrA-)rfbD1?recA1relA1?endA1spoT1?thi-1Δ(mcrC-mrr)114::IS10CommonlyusedfortiteringM13phagebecauseofthestrain'sF'plasmid,whichcarriesKanR,anditsslowgrowth,whichpromoteseasyvisualizationofplaques.HB101F-mcrBmrrhsdS20(rB-mB-)recA13leuB6ara-14proA2lacY1galK2xyl-5mtl-1rpsL20(SmR)glnV44λ-Pleasenotethatdifferentsourceshavedifferentgenotypessotreatthisinformationwithcaution.FromaGIBCOBRLlistofcompetentcells.HybridofE.coliK12andE.coliB(but98%KstrainAB266accordingtoSmithetal.)HostforpBR322andmanyplasmidsSigmaliststhedeletionΔ(gpt,proA).Checkthis.PromegadoesnotlistF-,mcrB,ormrrStreptomycinresistantReferences:Boyer,H.W.andRoulland-Dussoix,D.(1969)J.Mol.Biol.41,459.Smith,M.,Lorow,D.,andJessee,J.(1989)FOCUS11,56-pdfversionfromInvitrogenLacksSandGreenbergJR(1977)JMolBiol114:153.HMS174(DE3)F-recA1hsdR(rK12-mK12+)(DE3)(RifR)HMS174strainsprovidetherecAmutationinaK-12background.LikeBLR,thesestrainsmaystabilizecertaintargetgeneswhoseproductsmaycausethelossoftheDE3prophage.DE3indicatesthatthehostisalysogenoflDE3,andthereforecarriesachromosomalcopyoftheT7RNApolymerasegeneundercontrolofthelacUV5promoter.SuchstrainsaresuitableforproductionofproteinfromtargetgenesclonedinpETvectorsbyinductionwithIPTG.High-Control(tm)BL21(DE3)(Lucigen)F–ompTgaldcmhsdSB(rB-mB-)(DE3)/Mini-FlacIq1(Gentr)TheHI-ControlBL21(DE3)cellscontainasingle-copyBACplasmidharboringaspeciallyengineeredversionofthelacIq1repressorallele.ThelacIq1alleleexpresses~170-foldmorelacrepressorproteinthanthewild-typelacIgene.TheincreasedpooloflacrepressorinHI-ControlBL21(DE3)cellsmaintainstightcontrolovertheexpressionofT7RNApolymerasefromthelacUV5promoter,reducingleakyexpressionofgenesclonedunderaT7promoter.anE.coliBstrainwithDE3,aλprophagecarr