Boundaries of the Origin of Replication Creation of a pET-28a-Derived Vector with p15A Copy Control Allowing Compatible Coexistence with pET Vectors 英文参考文献.docVIP

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Boundaries of the Origin of Replication Creation of a pET-28a-Derived Vector with p15A Copy Control Allowing Compatible Coexistence with pET Vectors 英文参考文献.doc

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Boundaries of the Origin of Replication Creation of a pET-28a-Derived Vector with p15A Copy Control Allowing Compatible Coexistence with pET Vectors 英文参考文献

BoundariesoftheOriginofReplication:Creationof apET-28a-DerivedVectorwithp15ACopyControl AllowingCompatibleCoexistencewithpETVectors SarmithaSathiamoorthy1,JumiA.Shin1,2* 1DepartmentofChemistry,UniversityofToronto,Mississauga,Ontario,Canada,2InstituteofBiomaterialsandBiomedicalEngineering,UniversityofToronto,Toronto, Ontario,Canada Abstract During our studies involving protein-DNA interactions, we constructed plasmid pSAM to fulfill two requirements: 1) to facilitatetransferofclonedsequencesfromwidelyusedexpressionvectorpET-28a(+),and2)toprovideavectorcompatible with pBR322-derived plasmids for use in cells harboring two different plasmids. Vector pSAM is a pET-28a(+)-derived plasmidwiththep15Aoriginofreplication(ori);pET-28a(+)containsthepBR322repliconthatisincompatiblewithother pBR322-derivedplasmids.ByreplacingtheoriginalpET-28a(+)replicon–comprisingtheori,RNAI,RNAII,andRom–withthe p15A replicon, we generated pSAM, which contains the pET-28a(+) multiple cloning site and is now compatible with pBR322-derivedvectors.PlasmidcopynumberwasassessedusingquantitativePCR:pSAMcopynumberwasmaintainedat 1864copiespercell,consistentwiththatofotherp15A-typevectors.CompatibilitywithpBR322-derivedvectorswastested with pGEX-6p-1 and pSAM, which maintained their copy numbers of 49610 and 1464, respectively, when both were presentwithinthesamecell.Swappingoftheoriisacommonpractice;however,itisvitalthatallregionsoftheoriginal repliconberemoved.AdditionalvectorpSAMRNAIillustratedthatincompatibilityremainswhenportionsofthereplicon, suchasRNAIand/orRom,areretained;pSAMRNAI,whichcontainstheintactRNAIbutnotROM,loweredthecopynumber of pGEX-6p-1 to 1862 in doubly transformed cells due to retention of the pET-28a(+)-derived RNAI. Thus, pSAMRNAI is incompatiblewithvectorscontrolledbythepBR322repliconandfurtherdemonstratestheneedtoremoveallportionsof the original replicon and to quantitatively assess copy number, both individually and in combination, to ensure vector compatibility. To our kn