• Introduction
• Genotypeinformation
• Whyendo-freeplasmidprepisnotthebestmethod
• PlasmidproductionwithClearColi
• Endotoxicity/LALlevels
• TransfectionandproteinexpressionfromClearColiPlasmids
• GrowthratesforClearColiK-12cells
• Usefulreferencearticles
• ClearColilicensinginformation

IntroductiontoClearColi®technology:

Isthereabetterwaytoeliminateendotoxincontamination?
Nowthereis. 

Insteadofremovinglipopolysaccharide(LPS)contaminationfromyourproteinorplasmidDNApreparations,eliminatetheLPSatthesource. GeneticallymodifiedLPSfromanovel E.coli strainproducesfunctionallycleanrecombinantproteinsandplasmids. ClearColi®cellsarethefirstcommerciallyavailablecompetentcellswithamodifiedLPS(LipidIV_A -seeFig.1)thatdoesnottriggertheendotoxicresponseinmammaliancells.ClearColicellslackoutermembraneagoNISTsforhTLR4/MD-2activation;therefore,activationofhTLR4/MD-2signallingbyClearColiisseveralordersofmagnitudelowercomparedwith E.coli wild-typecells,andplasmidDNApreparedfromClearColiisvirtuallyfreeofendotoxicactivity.AfterminimalpurificationfromClearColicells,proteinsorplasmids(whichmaystillcontain LipidIV_A)canstillbeusedinmostapplicationswithoutelicitinganendotoxicresponse(seeEndotoxicityLALLevelsfordetails).

Figure1.TheLPSofanormalE.colicellcomparedtothegeneticallymodifiedLipidIVAfromClearColicells. InClearColi,theoligosaccharidechainhasbeendeleted,andtwoofthesixacylchainshavebeenremovedtodisabletheendotoxinsignal.

ModificationstothegenotypeoftheClearColicellsconsistofsevenseparategenedeletions,therebyensuringthatthereisnochanceofgeneticreversionbacktowildtypeandproductionofnormalLPS. ThesemutationsresultinthedeletionoftheoligosaccharidechainfromtheLPS,makingiteasiertoremovetheresultinglipidIVA fromthedownstreamproduct. Moreimportantly,twoofthesixacylchainsaredeleted. ThesixacylchainsoftheLPSarethetriggerthatisrecognizedbytheToll-likereceptor4(TLR4)incomplexwithmyeloiddifferentiationfactor2(MD-2),causingactivationofNF-ƙBandproductionofproinflammatorycytokines. LipidIVA,whichcontainsonlyfouracylchains,isnotrecognizedbyTLR4andthusdoesnottriggertheendotoxicresponse(seeFig.2).

Fig.2.ComparisonofrelativeNF-κBinductioninHEK-BlueCellsusingpurifiedLPSfromaK-12 E.coli strainorfrompure,syntheticallymanufacturedLipidIVA.

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GenotypeInformation:

ClearColiK-12competentcellshavethefollowinggenotype:

F-,&lamBDa;- ΔendAΔrecAmsbA52frr181ΔgutQΔkdsDΔlpxLΔlpxMΔpagPΔlpxPΔeptA

Sevenspecificdeletionmutations(ΔgutQΔkdsDΔlpxLΔlpxMΔpagPΔlpxPΔeptA)encodethemodificationofLPStoLipidIV_A,whileoneadditionalcompensatingmutation(msbA52)enablesthecellstomaintainviABIlityinthepresenceoftheLPSprecursorlipidIV_A.

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WhyEndo-freePlasmidPrepisnottheBestMethod

Topreventtoxicityincellstobetransfected,plasmidsproducedin E.colimustbeessentiallyfreeofendotoxin.However,efficienteliminationofendotoxinisachallengingtask,andendo-freeplasmidprepmethodsareexpensiveandtimeconsuming. ClearColiK-12cellsproduceplasmidDNAwithendotoxinlevelslessthanorequaltoplasmidspreppedfromstandardE.colicloninglinesandQiagen'sEndofreeMaxiPrepkits. 

ClearColiK-12cellsallowuseofstandardplasmidprepinsteadofendo-freemethods:

• Savesupto90%inplasmidprepcosts
• Saves1hourormoreinpreptime
• Hightransfectionandproteinexpressionlevelswithoutconcernforendotoxincontamination

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PlasmidProductionwithClearColiK-12Cells

ClearColiK-12cellsareendA-andrecA-forthehighestqualityplasmidproduction. PlasmidyieldsfromClearColiK-12cellsareequalorgreaterthanthoseobtainedfromnormalDH10Bcompetentcells. Table1comparesyieldsfrom1mLminiprepsforbothClearColiK-12andE.Cloni10G(equivalentOD'swereusedforbothpreps). 

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Endotoxicity/LALLevels

Limulus amebocyteassaytestingisanFDA-approvedmethodfordetectionofendotoxinsandthemostcommonassayused. Asshowninfigure3,astandardplasmidpurificationstepforDNAproducedfromClearColicellsresultsinLALresponselevelslessthan1%ofthatproducedbyplasmidsderivedfromstandardDH10Bcellsandstandardprepmethods. TheEUlevelsdetectedfromClearColiK-12derivedplasmidsarealsoequivalentorlowerthantothoseobtainedfromDH10BderivedplasmidspreparedwithQiagen'sEndofreeMaxiprepkits(datanotshown).

Fig.3Comparisonofpost-plasmidpurificationendotoxinunitsdetectedfromClearColiK-12(redbars)andDH10B(E.cloni10G,greybars)competentcells.PlasmidDNAfromClearColidemonstratessignificantreductioninEU/mgwithouttheneedforendotoxinfreeplasmidprepkits.

ItshouldbenotedthattheresidualEUmeasurementsarelikelyduetothenon-specificnatureoftheLALassayunlessextraneousLPScontaminationfromothersourcesispresent.TheLALassayisactivatedsolelybythe4´-monophosphoryldiglucosaminebackboneofLPS. LALactivityisminimallyinfluencedbyacylationpatternofLPS,thekeydeterminantofendotoxicityineukaryoticcells. TheLALassayalsorecognizesawiderspectrumofLPS/lipidAvariantsthanthecentralcellularendotoxinsensorsystemofthehumanimmunecellsystem. Assuch,falsepositiveresultscanandwillresultduetothelackofspecificityoftheassay.

Alternativetoxicityassays,suchasthoseusingHEK-Bluecells(seeClearColi®BL21(DE3)cellsformoreinformation)suggestthateveninthepresenceofEUlevelsabovethreshholdsnormallytargetedbyresearchers,theactualimmunogeniceffectsfromClearColi-derivedproductsarenon-existent. Duetothenon-specificityoftheLALassaywhencombinedwithlipidIVA fromClearColi,itissuggestedthatresearchersconsideralternativemethodsofendotoxinmeasurement.

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TransfectionandproteinexpressionfromClearColiPlasmids

Withtheoriginalsourceofendotoxineliminated,itisnowpossIBLetotransfectplasmidDNApreppedwithstandardmethodsdirectlyintohumanorothermammaliancelllineswithoutconcernforcellviability,alteredcellularresponsesorpoorproteinexpression. Toprovethis,aplasmid(pME-HA)containingageneencodingafluorescentproteinwasclonedintobothClearColiK-12andDH10BE.coli. TheplasmidfromClearColiwasthenisolatedviastandardQiagenMaxiprepkitmethod,whiletheplasmidfromDH10BwasisolatedusingQiagen'sEndofreeMaxiKit. TheresultingplasmidsweretransfectedintoHEK293Tcellsforproteinexpression(Figure4).  Nodifferencesincellviabilityorproteinexpressionlevelshavebeenobservedwhenusinganon-endofreeplasmidprepmethodincombinationwithClearColi-derivedplasmids. 

Figure4.ComparisonofexpressionofagreenfluorescentproteininHEK293TcellsfromClearColi-derivedplasmidsandstandardmaxiprep(left)vs.DH10B-derivedplasmidsandendofreemaxiprep(right).Theupperpanelsshowfluorescence;thelowerpanelsshowacombinedfluorescenceandbrightfieldimage.

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GrowthRatesforClearColiK-12Cells

ClearColiK-12cellsgrowatapproximately50%oftherateofnormalDH10Bcells(seeFig.5). Usersshouldexpecttoseeverysmallcoloniesforthefirst24hoursafterplatingtransformants. Lucigenrecommendsincubatingplatesfor32-40hoursbeforepickingcoloniesforfutureexperiments. Whengrowntosufficientdensities,ClearColiK-12cellsproducesimilarplasmidyieldsasnormalDH10Bcells.

Figure5.ComparisonofgrowthratesforClearColiK12ElectrocompetentCellsvs.E.cloni 10GELITEElectrocompetentcells. CellsweretransformedwithpME-HA-CometandinoculatedtoaninitialOD600 of~0.01in200mLofLBMillermediumandgrownat37°Cwithshakingat210rpm.TheOD600 ofthecultureswasrecordedeveryhalfhour.

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RelevantReferenceArticles:

• Teghanemt,etal,MolecularBasisofReducedPotencyofUnderacylatedEndotoxins,JImmunol,2005;175:4669-4676
• Mamat,etal,SingleaminoacidsubstitutionsineitherYhjDorMsbAconferviabilityto3-deoxy-D-manno-oct-2-ulosonicacid-depletedEscherichiacoli,MolecularMicroBIOLOGy,2008,67(3),633–648
• Meredith,etal,RedefiningtheRequisiteLipopolysaccharideStructureinEscherichiacoli,ACSChemicalBiology,2006,1(1),33-42
• Brandenburg,etal,TheExpressionofEndotoxicActivityintheLimulusTestasComparedtoCytokineProductioninImmuneCells,CurrentMedicinalChemistry,2009,16,2653-2660
• Gutsmann,etal.StructuralprerequisitesforendotoxicactivityintheLimulustestascomparedtocytokineproductioninmononuclearcells,InnateImmunity,2010,16(1),39-47
• BeomSeokPark1etal.,ThestructuralbasisoflipopolysacchariderecognitionbytheTLR4–MD-2complex,Nature458,1191-1195(30April2009)

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ClearColiLicensingInformation:

ClearColiCompetentcellsaresubjecttoUSPatent8,303,964andotherUSandforeignpendingpatents.

LucigenCorporation("Lucigen")hasalicensefromResearchCorporationTechnologiestosellClearColicompetentcellstothird-partiesfornon-commercialresearchpurposesonly.Aseparatelicenseisrequiredforanycommercialuse.Formoreinformationabouttheuseofthisproductbycommercialentities,pleasereviewour fulllicensingpage.

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