Experimental Protocols in Biotechnology PDF
Preview Experimental Protocols in Biotechnology
Neelima Gupta Varsha Gupta Editors Experimental Protocols in Biotechnology S P H PRINGER ROTOCOLS ANDBOOKS Forfurther volumes: http://www.springer.com/series/8623 SpringerProtocols Handbooks collects adiverse range ofstep-by-steplaboratorymethods andprotocolsfromacrossthelifeandbiomedicalsciences.Eachprotocolisprovidedinthe Springer Protocol format: readily-reproducible in a step-by-step fashion. Each protocol openswithanintroductoryoverview,alistofthematerialsandreagentsneededtocomplete theexperiment,andisfollowedbyadetailedproceduresupportedbyahelpfulnotessection offeringtipsandtricksofthetradeaswellastroubleshootingadvice.Withafocusonlarge comprehensive protocol collections and an international authorship, Springer Protocols Handbooksareavaluableadditiontothelaboratory. Experimental Protocols in Biotechnology Edited by Neelima Gupta Chhatrapati Shahu Ji Maharaj University, Kanpur, Uttar Pradesh, India Varsha Gupta Department of Biotechnology, Chhatrapati Shahu Ji Maharaj University, Kanpur, Uttar Pradesh, India Editors NeelimaGupta VarshaGupta ChhatrapatiShahuJiMaharaj DepartmentofBiotechnology University ChhatrapatiShahuJiMaharajUniversity Kanpur,UttarPradesh,India Kanpur,UttarPradesh,India ISSN1949-2448 ISSN1949-2456 (electronic) SpringerProtocolsHandbooks ISBN978-1-0716-0606-3 ISBN978-1-0716-0607-0 (eBook) https://doi.org/10.1007/978-1-0716-0607-0 ©SpringerScience+BusinessMedia,LLC,partofSpringerNature2020 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLCpartofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Dedication Dedicatedtoallbiotechnologistswhostrivedhardduringtheirlivestoexploreanddevelop bio-techniquesfor thecauseofscienceandsociety Preface Thispublicationofprotocolmanualisanoutcomeofintenseworkshop“Nationalworkshop onAdvancedMolecularBiotechniques”conductedbysomeofthebestmindsinIndia.The approachthatsomeoftheresearcherstookintheirexperiments,alongwiththeirproblem- solvingstylewascommendableandwasappreciatedbythesciencefraternity. Lookingattheresponse,theorganizersdecidedtoextendthestandardizedprotocolsto alltheresearchersandscientistsworkinginsimilarfields.Thisexperimentalprotocolmanual coversmanyimportanttechnologieswhichareroutinelyusedinthelaboratoriesacrossthe world. Wehavemadeanattempttocomprehensivelycoverdifferentaspectsofvarioustechni- ques, starting from the introduction, the importance of the technique, areas where the technique can be utilized, principle, working, the requirements and standardized method- ology. This is different from other protocol series in its essence of showing the possible outcomesafterexperimentation.Italsohasnotesontroubleshootingthatexplainspossible precautionsandguidestheresearchersinseamlesslyexecutingtheexperiments. Theprotocolmanualstartswithmicrobialinfectiondetectioninplantswithelaboration of the technique. Chapter 1 has standardized protocols for screening and detection of Ralstonia solanacearum, (a Gram-negative phytopathogen) a causative agent of bacterial wiltdiseaseinSolanaceaecropsleadingtosevereagroeconomiclosses.Chapter2elaborates detection of Fasciola infection in livestock production. Elaborate protocols for antigen extraction and detection from stool are provided which gives an estimate about pathogenload. Insituimaging,bothinliveandfixedzebrafishembryosusingcytomegalovirus(CMV) promoter-driven expression of fusion proteins, is discussed in Chapter 3. The chapter presents protocols for plasmid selection and microinjection, screening of embryos having clonesexpressingthefusionproteins,immunostainingandimagingofzebrafishembryosto studylocalizationanddynamicsofproteinsofinterestencodedbyplasmidvectors. With growing population and restriction of usable agricultural areas, aquaculture and fisheries hold high prospects in future. Chapter 4 elaborates techniques to monitor hema- tological parameters to assess the health status for diagnosis and prevention of diseases in fish.Asthetechniquesofhematologyaredifferentinfishesandmammals,assessmentoffish bloodparametersisdemonstratedwithcompletedescriptionofthetechniquesandmethods employedfor theidentificationofbloodparasitesandhemeindices. Nematode isolation and detection are challenging. They are responsible for severe infectionsinanimalsandplants.InChapter5,electronmicroscopicexaminationprocedures alongwithmoleculartaxonomyofroundwormstosegregatetaxafromarthropod-parasitic, freshwater,marinewater,andsoilhabitatshavebeenelaborated. Chapter 6 focuses on various computational approaches in the process of drug discov- ery.Itcoversvariousmethodsandalgorithmsofcomputationalbiologywhichcanbeused for structural predictions of lead compound. The chapter not only covers screening and dockingmethodologiesbutalsoelaboratespharmacophoremappingwithdiscussionsabout metabolismandeliminationofdrugs. vii viii Preface Antibiotic resistance is posing challenge for scientific and medical fraternity. Chapter 7 dealswiththestudyofmicrobialbiofilms,theirdiversityaswellasgeneticcomplexitywhich leads to microbial resistance to biocontrol agents such as antimicrobial compounds. It elaborates effective and safe antibacterial alternative strategies as using bacteriophages in aneraofrapidlyemergingantibioticresistance. Chapter8elaboratesnon-radioisotopicsafeandeffectiveprotocolsforstudyingrevers- iblephosphorylationanddephosphorylationreactionsinplantsystem.Theprotocolscanbe extendedtoanyotherexperimentalsystem.Detailedmethodologyforinvivophosphoryla- tiondetectionisdiscussedwithstandardizedprotocolsusingwesternblottingandprobing byanti-phosphoantibodies. Chapter9elaboratesisolationofplasmamembraneandtonoplast-enrichedfractions.It provides standardized methodology for estimation of ATPase hydrolytic activity with an importantroleofpossible inhibitoryinfluencesofmoleculeswhichobstructsitshydrolytic activity. Chapter 10 explains detection, quantification, and annotation of microRNAs (miR- NAs). The conserved miRNA family members play an important function in a number of physiologicalprocesseswithanessentialroleinthecontrolofgeneexpression. Cell analysis and identification have become easier with advancement in technology. Chapter 11 covers working protocols for isolation, purification, and characterization of naturalkillercells(NKcells)frommousespleenandbonemarrow. Powerful technologies of detection of gene expression which have completely over- shadowed northern blotting are discussed in Chapters 12 and 13. Chapter 12 elaborates RNA isolation, quality and quantity evaluation, reverse transcription, and real-time PCR reaction with example of quantification and normalization of gene expression. Chapter 13 elaboratesversatileandemergingtechnologyofdigitaldropletPCRanditsapplications. Understanding enzymes, its kinetics, determination of V and the role of inhibitor max have always been challenging for researchers. Chapter 14 brings standardized protocol of alkaline phosphatase activity determination and gradually guides researcher to determine V ,K ,influenceofinhibitorsandpositive-negativefeedbackloops. max M Lastly, Chapter 15 deals with the introduction and evaluation of non-conserved long non-coding RNA (LncRNA). LncRNA is increasingly finding importance as an important regulatorofgeneexpression.HeredetectionoflncRNAfromtissuesectioniselaborated. Theprotocolscompiledareready-to-usemethodswhicharestandardizedfromexisting protocols. We hope that the researchers will find these protocols useful and will be able to tapyearsofexperienceofresearchersthroughthisbook. Kanpur,India NeelimaGupta Kanpur,India VarshaGupta Acknowledgements Wewould liketo startbypraisingand thanking theAlmightyforgiving us theinspiration, guidingusthroughtheentirejourney,andmakingtheexperiencememorable. Weexpressourheartfeltandsinceregratitudetoeveryonewhohascontributedselflessly tothegrowthanddevelopmentofscience.Unfailingpositivespiritdespitethefailuresand strugglesofthescientistsallover theworldhaskeptthequestforknowledgealiveandlaid thefoundationstoneaswellaspavedourpathintothetechnologicalworld.Reflectionsof their knowledge in writings, books, publications, and other documents have helped and inspiredustocapturesomeoftheknowledgeandpassitontoreaders. We also want to express our gratitude to our parents for their love, blessings, and continuousselflesssupport,whichhavemadeuswhatwearetoday,andtooursiblingsfor providingcontinuousencouragementandsupport.ThanksareduetoMeghanaandAgastya whospenttheir timeandhelpeduswithwritingsanddiagrams. Last but not least, we would like to thank all the colleagues, friends, and students for their support and help during the preparations. We sincerely acknowledge the untiring support of the entire Springer team for their cooperation and support. Special mention must be made to Ms. Aakanksha Tyagi for taking all the trouble to help us whenever required. Her continuous guidance and support made this possible. We would also like to thankMr.JohnMartynandMs.Padmapriyaforallthecooperationandsupport.Thanksto ourspousesfortheirimmensepatienceandbeingaconstantsourceofinspirationthrough- outthejourney. ix Contents Dedication.................................................................. v Preface ..................................................................... vii Acknowledgements............................................................ ix EditorsandContributors ...................................................... xiii 1 Enzyme-LinkedImmunosorbentAssayDetectionofBacterialWilt–Causing Ralstoniasolanacearum.................................................. 1 PramilaDeviUmrao,VineetKumar,andShilpaDeshpandeKaistha 2 DiagnosisofTropicalFascioliasisUsingSpecificAntigenDetectioninFeces ofInfectedHumanBeingorAnimalSpecies ............................... 19 NeelimaGupta,SaidI.Shalaby,DileepK.Gupta,andMonaA.Awad 3 CMVPromoter-DrivenExpressionandVisualizationofTaggedProteinsin LiveandFixedZebrafishEmbryonicEpidermis............................. 29 KirtiGuptaandMahendraSonawane 4 DetectionofBloodParasitesandEstimationofHematologicalIndices inFish................................................................. 43 NeelimaGuptaandShahlaNigar 5 TechniquestoConductMorphologicalandMolecularInvestigations onNematodes.......................................................... 75 AashaRana,AnshikaYadav,AashaqHussainBhat,AshokKumar Chaubey,andSandeepK.Malhotra 6 ComputationalApproachesinDrugDesigningandTheirApplications........ 95 DevBukhshSinghandRajeshKumarPathak 7 BacteriophageControlforPseudomonasaeruginosaBiofilmFormation andEradication......................................................... 119 PramilaDeviUmrao,VineetKumar,SadhanaSinghSagar,and ShilpaDeshpandeKaistha 8 DetectionofPhosphoproteins(PhosphoserineandPhosphothreonine) fromThylakoidMembranesUsingWesternBlotting ........................ 139 VarshaGuptaandBaishnabCharanTripathy 9 EstimationofPlasmaMembraneandTonoplastATPaseActivityin PlantTissues ........................................................... 155 NeerjaSrivastava 10 QuantificationofConservedMicroRNAinPlantsandValidationof NewTargets ........................................................... 163 LataIsraniShukla 11 IsolationofNaturalKillerCellsfromMouseBoneMarrowandSpleen........ 183 AsmitaDas xi
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