An Optimized, Chemically Regulated Gene Expression System for Chlamydomonas 英文参考文献.docVIP

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An Optimized, Chemically Regulated Gene Expression System for Chlamydomonas 英文参考文献.doc

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An Optimized, Chemically Regulated Gene Expression System for Chlamydomonas 英文参考文献

AnOptimized,ChemicallyRegulatedGeneExpression SystemforChlamydomonas PaolaFerrante1,ClaudiaCatalanotti1,GiuliaBonente2,GiovanniGiuliano1* 1ItalianNationalAgencyforNewTechnologies,EnergyandtheEnvironment(ENEA),CasacciaResearchCenter,Rome,Italy, 2DipartimentoScientificoeTecnologico, UniversityofVerona,Verona,Italy Abstract Background: Chlamydomonas reinhardtii is a model system for algal and cell biology and is used for biotechnological applications, such as molecular farming or biological hydrogen production. The Chlamydomonas metal-responsive CYC6 promoterisrepressedbycopperandinducedbynickelions.However,inductionbynickelisweakinsomestrains,poorly reversiblebychelatingagentslikeEDTA,andcauses,athighconcentrations,toxicitysideeffectsonChlamydomonasgrowth. Removalofthesebottleneckswillencouragethewideuseofthispromoterasachemicallyregulatedgeneexpressionsystem. Methodology:Usingacodon-optimizedRenillaluciferaseasareportergene,weexploredseveralstrategiestoimprovethe strengthandreversibilityofCYC6promoterinduction.UseofthefirstintronoftheRBCS2geneorofamodifiedTAPmedium increasesthestrengthofCYC6inductionupto20-fold.Inthemodifiedmedium,inductionisalsoobtainedafteradditionof specific copper chelators, like TETA. At low concentrations (up to 10mM) TETA is a more efficient inducer than Ni, which becomes a very efficient inducer at higher concentrations (50mM). Neither TETA nor Ni show toxicity effects at the concentrationsused.UnlikeinductionbyNi,inductionbyTETAiscompletelyreversiblebymicromolarcopperconcentrations, thusresultinginatransient‘‘wave’’inluciferaseactivity,whichcanberepeatedinsubsequentgrowthcycles. Conclusions: We have worked out a chemically regulated gene expression system that can be finely tuned to produce temporally controlled ‘‘waves’’ in gene expression. The use of cassettes containing the CYC6 promoter, and of modified growthmedia,isareliableandeconomicallysustainablesystemforthetemporallycontrolledexpressionofforeigngenesin Chlamydomonas. C