Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp. PCC 6803 英文参考文献.docVIP

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Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp. PCC 6803 英文参考文献.doc

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Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp. PCC 6803 英文参考文献

EthyleneSynthesisandRegulatedExpressionof RecombinantProteininSynechocystissp.PCC6803 FernandoGuerrero,Vero′nicaCarbonell.,MatteoCossu.,DaniloCorreddu,PatrikR.Jones* UniversityofTurku,DepartmentofBiochemistryandFoodChemistry,Turku,Finland Abstract The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detectedintheheadspaceofclosedcultures.Asyntheticcodon-optimizedgeneencodingN-terminalHis-taggedEFE(EFEh) was expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse promotersinaself-replicatingbroadhost-rangeplasmid.Ethylenesynthesiswasstablymaintainedinbothorganismsin contrasttoearlierworkinSynechococcuselongatusPCC7942.Therateofethyleneaccumulationwasusedasareporterfor protein expression in order to assess promoter strength and inducibility with the different expression systems. Several metal-induciblecyanobacterialpromotersdidnotfunctioninE.colibutwerewell-regulatedincyanobacteria,albeitatalow levelofexpression.TheE.colipromoterPtrcresultedinconstitutiveexpressionincyanobacteriaregardlessofwhetherIPTG was added or not. In contrast, a Lac promoter variant, PA1lacO-1, induced EFE-expression in Synechocystis at a level of expressionashighastheTrcpromoterandallowedafinelevelofIPTG-dependentregulationofprotein-expression.The regulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing promotersystemwasalsoconstructedandshowntofunctionwellinE.coli,however,onlyaverylowlevelofEFE-activity wasobservedinSynechocystis,independentofcelldensity. Citation:GuerreroF,CarbonellV,CossuM,CorredduD,JonesPR(2012)EthyleneSynthesisandRegulatedExpressionofRecombinantProteininSynechocystis sp.PCC6803.PLoSONE7(11):e50470.doi:10.1371/journal.pone.0050470 Editor:BrettNeilan,UniversityofNewSouthWales,Australia ReceivedJuly26,2012;AcceptedOctober23,2012;PublishedNovember21,2012 Copyright: ? 2012 Guerrero et al. This is