An Effective Strategy for a Whole-Cell Biosensor Based on Putative Effector Interaction Site of the Regulatory DmpR Protein 英文参考文献.docVIP

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An Effective Strategy for a Whole-Cell Biosensor Based on Putative Effector Interaction Site of the Regulatory DmpR Protein 英文参考文献.doc

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An Effective Strategy for a Whole-Cell Biosensor Based on Putative Effector Interaction Site of the Regulatory DmpR Protein 英文参考文献

AnEffectiveStrategyforaWhole-CellBiosensorBased onPutativeEffectorInteractionSiteoftheRegulatory DmpRProtein SaurabhGupta1,3,MritunjaySaxena1,NeeruSaini1,Mahmooduzzafar3,RitaKumar1*,AnilKumar2* 1Institute of Genomics and Integrative Biology, Mall Road, Delhi, India, 2National Institute of Immunology, New Delhi, India, 3Jamia Hamdard University, Hamdard Nagar,NewDelhi,India Abstract IntroductionandRationale:Thedetectionofbioavailablephenolisaveryimportantissueinenvironmentalandhuman hazardassessment.Despitemodestdevelopmentsrecently,thereisasternneedfordevelopmentofnovelbiosensorswith highsensitivityforpriorityphenolpollutants.DmpR(Dimethylphenolregulatoryprotein),anNtrC-likeregulatoryprotein forthephenoldegradationofPseudomonassp.strainCF600,representsanattractivebiosensorregimen.Thus,wesought todesignanovelbiosensorbymodifyingthephenoldetectioncapacityofDmpRbyusingmutagenicPCR. Methods:Bindingsitesof‘A’domainofDmpRwerepredictedbyLIGSITE,andmoleculardockingwasperformedbyusing GOLDtoidentifytheregionswherephenolmayinteractwithDmpR.Totalfivepointmutations,onesingleatposition42 (Phe-to-Leu),twodoubleat140(Asp-to-Glu)and143(Gln-to-Leu),andtwodoubleatL113M(Leu-to-Met)andD116A(Asp- to-Ala)werecreatedinDmpRbysite-directedmutagenesistoconstructthereporterplasmidspRLuc42R,pRLuc140p143R, andpRLuc113p116R,respectively.Luciferaseassayswereperformedtomeasuretheactivityoflucgeneinthepresenceof phenolanditsderivatives,whileRT-PCRwasusedtochecktheexpressionoflucgeneinthepresenceofphenol. Results: Only pRLuc42R and pRLuc113p116R showed positive responses to phenolic effectors. The lowest detectable concentration of phenol was 0.5mM (0.047mg/L), 0.1mM for 2, 4-dimethylphenol and 2-nitrophenol, 10mM for 2, 4, 6- trichlorophenol and 2-chlorophenol, 100mM for 2, 4-dichlorophenol, 0.01mM for 4-nitrophenol, and 1mM for o-cresol. Theseconcentrationsweremeasuredbymodified luciferaseassay within3hrs comparedto6–7hrsinprevious studies. Importantly,increasedexpressionofluciferasegeneof