Ribosomal Binding Site Switching An Effective Strategy for High-Throughput Cloning Constructions 英文参考文献.docVIP

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Ribosomal Binding Site Switching An Effective Strategy for High-Throughput Cloning Constructions 英文参考文献.doc

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Ribosomal Binding Site Switching An Effective Strategy for High-Throughput Cloning Constructions 英文参考文献

RibosomalBindingSiteSwitching:AnEffectiveStrategy forHigh-ThroughputCloningConstructions YangboHu1.,LipengFeng1,2.,YunlongLi1,2,YongZhang1,PeiLu1,SimonRayner1,ShiyunChen1* 1KeyLaboratoryofAgriculturalandEnvironmentalMicrobiology,WuhanInstituteofVirology,ChineseAcademyofSciences,Wuhan,China,2GraduateUniversityofthe ChineseAcademyofSciences,Beijing,China Abstract DirectcloningofPCRfragmentsbyTAcloningorbluntendligationaretwosimplemethodswhichwouldgreatlybenefit high-throughput(HTP)cloningconstructionsiftheefficiencycanbeimproved.Inthisstudy,wehavedevelopedaribosomal binding site (RBS) switching strategy for direct cloning of PCR fragments. RBS is an A/G rich region upstream of the translational start codon andis essential forgene expression. Change fromA/G toT/C in theRBS blocks itsactivity and therebyabolishesgeneexpression.Basedonthisproperty,weintroducedaninactiveRBSupstreamofaselectablemarker gene,anddesignedafragmentinsertionsitewithinthisinactiveRBS.Forwardandreverseinsertionsofspecificallytailed fragments will respectively form an active and inactive RBS, thus all background from vector self-ligation and fragment reverseinsertionswillbeeliminatedduetothenon-expressionofthemarkergene.TheeffectivenessofourstrategyforTA cloningandbluntendligationareconfirmed.Applicationofthisstrategytogeneover-expression,abacterialtwo-hybrid system, a bacterial one-hybrid system, and promoter bank construction are also verified. The advantages of this simple procedure,togetherwithitslowcostandhighefficiency,makesourstrategyextremelyusefulinHTPcloningconstructions. Citation:HuY,FengL,LiY,ZhangY,LuP,etal.(2012)RibosomalBindingSiteSwitching:AnEffectiveStrategyforHigh-ThroughputCloningConstructions.PLoS ONE7(11):e50142.doi:10.1371/journal.pone.0050142 Editor:BaochuanLin,NavalResearchLaboratory,UnitedStatesofAmerica ReceivedSeptember12,2012;AcceptedOctober17,2012;PublishedNovember21,2012 Copyright:?2012Huetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttri