Experimental Protocols in Biotechnology

252 Pages·2020·7.688 MB·English

by  Neelima Gupta, Varsha Gupta

Neelima Gupta
Varsha Gupta
Editors
Experimental
Protocols in
Biotechnology
S P H
PRINGER ROTOCOLS ANDBOOKS
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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
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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
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