- Savetime!Enzyme-freecloninginsecondswithcloning-readyvectorandcells.
- Highefficiency:›90%recombinants.
- Tightly-controlledexpressionofN-orC-terminal6xHistaggedproteins.
AlsoavailablewithcleavableSUMOSolubilityTag
CompleteCloningandExpressionSystem
TheExpressoT7CloningandProteinExpressionSystemsaredesignedforfast,easy,andefficientdirectionalcloningandexpressionofPCR-amplifiedgenes.Thesystemsarecompletewithpre-processedpETite™N-orC-Hiscloningvectors,andtwocompetentcelllines,suppliedinsingletransformationvials.HighefficiencyHI-Control™10GChemicallyCompetentCellsenablestablecloningandHI-ControlBL21(DE3)CompetentCellsprovidetightlycontrolledproteinexpression,thushelpingyouavoidexpressionproblemsseenwithleakyT7promoter-basedsystems.TheN-orC-terminal6xHistaggedproteinscanberapidlypurifiedusingstandardNichromatography.SystemsarealsoavailablethatencodeanN-terminal6xHisSUMOtagfusionforimprovedsolubilityofexpressedprotein.OtherExpressoSystemsareavailablewhichexpressproteinsunderthecontroloftheRhamnosepromoter.
Five-SecondDirectionalCloningofPCR-AmplifiedGenes
TheExpressoT7CloningandProteinExpressionSystemusesExpressioneeringTechnology™,anenzyme-freerecombinationalcloningstrategytoseamlesslyintegratethegenewiththevector.ThetargetgeneisamplifiedbyPCRusingprimersthatadd18base-pairsofvector-complementarysequencetobothendsofthegene.Unlikeotherrestrictionenzymebasedmethodsorligase-freecloningmethods,nofurthercleanuporenzymatictreatmentofthePCRproductisnecessary.Simplymix1µloftheunpurifiedPCRreactionwiththesuppliedpre-processedpETite™T7expressionvector,andtransformimmediatelyintotheHI-Control™10GChemicallyCompetentCellsprovided(Figure1).
Cloning-ReadyVectorsSaveHoursofResearchTime.
NewpETiteT7vectorsinclude:
- StrongT7promoterforhigh-levelexpression.
- ChoiceofN-terminalorC-terminal6xHisfusiontagsforfastproteinpurification.
- Smallsize(2.2kb)foreasierdownstreammanipulation.
- PatentedCloneSmart®technologyincreasescloningefficiency.
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Figure2.pETiteT7expressionvectors:Smallsize(2.2kbvs.5.4kbforpET)foreasiermanipulation,includingtargetedmutagenesis.Vectorsarepre-processedforinstantenzyme-freecloningofPCRproductswithachoiceofamino-terminalorcarboxyl-terminalfusionof6xHistagtoproteinofinterest. |
HighcloningefficiencywithHI-Control10GChemicallyCompetentCells.
ThehightransformationefficiencyofHI-Control10GChemicallyCompetentCellsensuresrecoveryofcloneswithprecisejunctionsandthecorrectorientation.Formostgenes,>90%ofcolonieswillhavethetargetgeneinsertedinthecorrectorientation.(Figure3).
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Figure3.HighcloningefficiencywithExpressoT7System. |
HI-ControlBL21(DE3)CellsControlLeakyProteinExpression
HI-ControlBL21(DE3)CellscontainhighlevelsoflacrepressortomaintaintightcontroloverexpressionofT7RNApolymerase.Tightercontrolmeansbettertoleranceofpotentiallytoxicgeneproducts(Figure4).
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Figure4.ExpressionofvariousproteinsusingtheExpressoT7SystemversusapETVector.GenesencodingaDNApolymerase,abluefluorescentprotein(BFP),orATPsynthasebsubunit(membraneprotein)wereclonedintopET28aorpETitevectorswithN-terminalorC-terminal6xHistags(asindicated).pET28aclonesweretransformedintostandardBL21(DE3)cells,andpETiteclonesweretransformedintoHI-ControlBL21(DE3)cellsforexpression.CulturesweregrowninLBat37°CtoanOD600of0.5to0.7(odd-numberedlanes)andinducedfor3hourswith1mMIPTG(even-numberedlanes).CellswerepelletedandlyseddirectlyinSDS-PAGEloADIngbuffer,and0.05ODequivalentswereanalyzedbygelelectrophoresis.ThegelwasstainedwithCoomassieblue. |
PurificationofActiveSolubleFluorescentProteinUsing6xHisTag
TheN-orC-terminal6xHistaggedproteinsexpressedusingtheExpressoT7CloningandExpressionSystemcanberapidlyaffinity-purifiedovercommerciallyavailablenickelresins.Anexampleofthepurificationofa6xHistaggedyellowfluorescentproteinclonedandexpressedusingtheExpressoT7SystemisshowninFigure5.
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Figure5.Purificationofa6xHistaggedfluorescentprotein.HI-ControlBL21(DE3)cellsharboringpETiteC-Hisvectorcontainingayellowfluorescentprotein(YFP)geneweregrownat37°CinLBmediatoanOD600of0.6(lane1),theninducedwith |
ImportantProductUseInformation:
ThisproductisthesubjectofU.S.Patent#6,709,861.AdditionalpatentapplicationsownedbyLucigenCorporationarepending.
The6xHistagislicensedfromHoffmann-LaRoche,Inc.,Nutley,NJand/orHoffmann-LaRocheLtd.,Basel,Switzerlandandisprovidedonlyfortheuseinresearch. InformationaboutlicensesforcommercialuseisavailablefromQiagenGmbH,QIAGENStrasse1,D-40724Hilden,Germany.Purificationofthe6xHistaggedproteinswithNi-NTAmanufacturedbyQIAGENishighlyrecommendedforbestperformancesandtoavoidpoorpurificationresults.
ORDERINFORMATION
TheExpressoT7Cloning&ExpressionSystemcontainspre-processedpETite®N-Hisand/orpETiteC-HisVectorDNA,HI-Control™10GChemicallyCompetentCellsforcloning,andHI-ControlBL21(DE3)ChemicallyCompetentCellsforproteinexpression.AlsoincludedareN-Hisand/orC-HisPositiveControlInsertDNAs,andtransformationpositivecontrolpUCDNA.TheExpressoT7SUMOCloningandExpressionSystemcontainspre-processedpETite®N-HisSUMOVectorDNA,HI-Control™10GChemicallyCompetentCellsforcloning,andHIControlBL21(DE3)ChemicallyCompetentCellsforproteinexpression.AlsoincludedareSUMOPositiveControlCInsertDNA,transformationpositivecontrolpUCDNA,SUMOExpressProtease,SUMOCleavageControlProtein,andforwardandreversePCRprimerstoconfirmclones.
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但严格来说，还是有区别的。因为基因的表达产物包括蛋白质和RNA（rRNA和tRNA）。如果产物是蛋白质，就是转录翻译；如果产物是RNA，就只是转录过程，没有翻译过程。
翻译是指mRNA在核糖体的帮助下翻译成肽链
基因表达则是二者的统一，即DNA转录成RNA，RNA翻译成肽链，并最终折叠成有意义的蛋白质
基因表达谱测序是直接对某一物种或特定细胞在某一功能状态下产生的mRNA进行高通量测序，可以用来研究基因的表达差异情况。该技术结合了转录组测序建库的实验方法，与转录组测序相比，基因表达谱测序要求的读长更短，测序通量更小，但仅可用于基因表达差异的研究。
转录组测序是RNA水平测序，相当于DNA水平的基因组测序，是一个框架。表达谱主要研究的是基因表达量的变化，上调或下降。先要有转录组或是基因组才可以做表达谱，否则没有Ref做参考。
转录组测序和表达谱测序其实都是通过高通量测序技术进行的，转录组测序主要是针对没有参考基因组（即基因组未完成测序）的物种，侧重于获得你材料的全部转录组信息；而表达谱则侧重于检测各个基因的表达量。
遗传物质的表达的产物是：蛋白质
DNA转录的产物是：mRNA
希望对你有帮助~
