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Basic Biotechniques for Bioprocess and Bioentrepreneurship This page intentionally left blank Basic Biotechniques for Bioprocess and Bioentrepreneurship Edited By Arvind Kumar Bhatt Department of Biotechnology, Himachal Pradesh University, India Ravi Kant Bhatia Department of Biotechnology, Himachal Pradesh University, India Tek Chand Bhalla Department of Biotechnology, Himachal Pradesh University, India AcademicPressisanimprintofElsevier 125LondonWall,LondonEC2Y5AS,UnitedKingdom 525BStreet,Suite1650,SanDiego,CA92101,UnitedStates 50HampshireStreet,5thFloor,Cambridge,MA02139,UnitedStates TheBoulevard,LangfordLane,Kidlington,OxfordOX51GB,UnitedKingdom Copyright©2023ElsevierInc.Allrightsreserved. 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Practitionersandresearchersmustalwaysrelyontheirownexperienceandknowledgeinevaluatingandusing anyinformation,methods,compounds,orexperimentsdescribedherein.Inusingsuchinformationor methodstheyshouldbemindfuloftheirownsafetyandthesafetyofothers,includingpartiesforwhomtheyhave aprofessionalresponsibility. Tothefullestextentofthelaw,neitherthePublishernortheauthors,contributors,oreditors,assumeany liabilityforanyinjuryand/ordamagetopersonsorpropertyasamatterofproductsliability,negligenceor otherwise,orfromanyuseoroperationofanymethods,products,instructions,orideascontainedinthe materialherein. ISBN:978-0-12-816109-8 ForinformationonallAcademicPresspublications visitourwebsiteathttps://www.elsevier.com/books-and-journals Publisher:StacyMasucci AcquisitionsEditor:LindaVersteeg-Buschman EditorialProjectManager:TimothyJ.Bennett ProductionProjectManager:NiranjanBhaskaran Coverdesigner:MilesHitchen TypesetbySTRAIVE,India Contents Contributors xv 7.5 Cultivationofanaerobic Foreword xix microorganisms 16 Preface xxi 7.6 Intracellularbacterialculture 17 8. Conclusion 17 References 17 Part I Isolation, screening and culture 2. Screening strategies maintenance ChayanikaPutatunda,PreetiSolanki, 1. Isolation of microorganisms 1 ShrutiPathania,AnilKumar,and AbhishekWalia MahinderKumarGupta 1. Introduction 23 1. Introduction 3 2. Conventionalstrainscreening 2. Clinicallyimportantmicroorganisms 3 techniques 24 2.1 Siteforisolation 3 2.1 Culture-dependentmethods 24 2.2 Sampling 4 2.2 Conventionalscreeningof 3. Agriculturallyimportantmicroorganisms 4 antimicrobials 24 3.1 Preliminaryassessment 9 3. Alternativecultivationmethods 25 3.2 Identificationofsitesandsample 4. Molecularmethodofmicrobialstrain collection 9 screeningstrategies 26 3.3 Storageandpretreatmentofsoil 4.1 Randomlyamplifiedpolymorphic samples 9 DNA 27 4. Dairyrelatedmicroorganisms 10 4.2 Restrictionfragmentlength 4.1 Samplecollection 10 polymorphism 29 4.2 Preservationofsamples 10 4.3 Pulsefieldgelelectrophoresis 29 5. Extremophiles 11 4.4 Amplifiedfragmentlength 5.1 Thermophiles 11 polymorphism 29 5.2 Psychrophiles 11 4.5 RioPrinter 30 5.3 Alkaliphiles/acidophiles 11 4.6 Multilocussequencetyping 30 5.4 Piezophiles 12 4.7 MALDI-TOF 31 5.5 Radiophiles 12 5. High-throughputscreeningtechniques 31 5.6 Xerophiles 12 5.1 AdvantagesofHTStechnology 32 5.7 Metallophiles 13 5.2 Analyticalmeasurement 32 5.8 Halophiles 13 6. “Omics”-basedscreeningtechniques 33 5.9 Microaerophiles 13 7. Virtualscreeningstrategies 36 6. Transportofsamples 13 8. Somepotentialapplicationofnovel 7. Industriallyimportantmicroorganisms 14 screeningstrategies 40 7.1 Sourcesofmicroorganisms 14 8.1 Inmedicineanddrugdiscovery 40 7.2 Enrichmentandisolation 14 8.2 Environmentalapplications 40 7.3 Cultivationofmicroorganisms 14 9. Conclusionsandfutureperspectives 41 7.4 Cultivationofextremophiles 16 References 41 v vi Contents 3. Identification, morphological, 4. Microbial activity and productivity biochemical, and genetic enhancement strategies characterization of ShashiKantBhatia,VijayKumar, microorganisms VirenderKumar,RaviKantBhatia, andYung-HunYang NiveditaSharma,NishaSharma, ShakshiSharma,PushpinderSharma,and 1. Introduction 85 BinduDevi 2. Isolationofmicrobes 85 1. Introduction 47 3. Statisticaldesignforcultureandreaction 2. Isolationofmicroorganisms 47 conditionoptimization 86 2.1 Methodsofisolation 47 4. Inductionstrategy 87 3. Identificationofmicrobes 52 5. Immobilization 88 3.1 Principlesoftaxonomy 52 5.1 Adsorption 90 3.2 Strategiesusedtoidentify 5.2 Covalentbinding 91 microbes 53 5.3 Entrapment 91 3.3 Morphologyofbacteria 59 5.4 Cross-linking 91 3.4 Morphologyoffungi 61 5.5 Othermethodsofimmobilization 92 4. Biochemicalcharacterizationof 6. Mutagenesisforenhancementof microbes 63 enzymeactivityandproductivity 92 4.1 Indoletest 63 6.1 Physicalandchemicalmutagenesis 93 4.2 Methylredtest 63 6.2 Directedevolution 95 4.3 VogesProskauertest 63 6.3 Sitedirectedmutagenesis 95 4.4 Citratetest 65 7. Metabolicengineering 95 4.5 Triplesugar-iron(TSI)agar 7.1 Improvementofmicrobesfor test 65 utilizationofcarbonsource 96 4.6 Carbohydratefermentationtest 67 7.2 Constructionofnewmetabolic 4.7 Oxidativefermentative(O-F) pathway 96 test 68 7.3 Increasedcofactorproductionand 4.8 Aminoaciddecarboxylasetest 69 regeneration 96 4.9 Litmusmilktest 69 7.4 Improvementofrobustnessto 4.10 Hydrogensulfidetest 70 stress 98 4.11 O-nitrophenyl-β-D-galactopyranoside 8. Co-culturestrategy 98 (ONPG)test 70 9. Conclusion 99 4.12 Phenylalaninedeaminasetest 72 Acknowledgment 99 4.13 Catalasetest 72 References 99 4.14 Oxidasetest 73 4.15 Gelatinhydrolysistest 73 5. Culture maintenance, 4.16 Starchhydrolysistest 74 preservation, and strain 4.17 Lipidhydrolysistest 74 improvement 4.18 DNAhydrolysistestor AmanKumar,SrijanaMukhia,AnilKumar, deoxyribonuclease(DNase)test 74 KiranDindhoria,NehaBaliyan,and 4.19 Coagulasetest 75 RakshakKumar 5. Geneticcharacterizationof microorganisms 75 1. Introduction 105 5.1 Microorganismswhosestudyis 2. Culturemediafordifferent aspects 106 2.1 Classificationbyphysicalnature 106 encompassedbymicrobial 2.2 Classificationbychemical genetics 75 composition 106 5.2 DeterminationofDNA 2.3 Classificationbypurpose/ sequences 76 functionaluse 107 5.3 Microbialfingerprinting 3. Sterilizationtechniques 108 methods 79 6. Conclusion 82 3.1 Heatsterilization 108 References 82 3.2 Gassterilization 110 Contents vii 3.3 Sterilizationbyradiation 110 3.2 Materialusedinrotorconstruction 138 3.4 Filtersterilization 110 3.3 Varioustypesofcentrifugation 4. Maintenanceandpreservationofpure technique 138 cultures 111 3.4 Typesofcentrifuges 138 4.1 Metabolicallyactivemethods 112 3.5 Separationmethodsindifferenttypes 4.2 Metabolicallyinactivemethods 112 ofcentrifugation 139 4.3 Microbialculturecollections 114 3.6 Applicationsofcentrifugation 5. Strainimprovement 114 techniques 142 5.1 Characteristicsofanimprovedstrain 116 3.7 Careofcentrifugationequipment 142 5.2 Methodsformicrobialstrain 3.8 Safetyaspectswhileoperatinga improvement 117 centrifuge 142 6. Conclusion 120 References 142 Acknowledgment 120 References 120 8. Spectroscopy—Principle, types, and applications Part II SwetaSinha,ChristineJeyaseelan, Laboratory techniques & GunjanSingh,TanyaMunjal,and DebaratiPaul instrumentation 1. Introduction 145 6. Biomolecules: Types, 2. Generaltypesofspectra 145 homogenization, bead beater, 2.1 Continuousspectra 145 2.2 Discretespectra 145 sonication 3. Principleofspectroscopy 146 ArshadJawed 3.1 Opticalinstrumentsinspectroscopy 147 3.2 Howspectroscopydifferentfrom 1. Introduction 125 spectrometry 147 2. Classificationofcelldisruption 4. Typesofspectroscopy 147 processes 126 4.1 Ultravioletandvisiblespectroscopy 147 2.1 Physicaldisruptionmethods 127 4.2 Infraredspectroscopy 150 2.2 Chemicaldisruption 127 4.3 Massspectrometry 155 2.3 Large-scalecelldisruption: 4.4 Nuclearmagneticresonance(NMR) Thebeadmill 128 spectroscopy 158 3. Conclusion 130 5. Conclusion 162 References 130 References 162 7. Centrifugation: Basic principle, 9. Protein purification: Basic types principles and techniques GauravSood,MinakshiSharma, AllaSingh,KrishanKumar, andRajeshKaushal DharamPaulChaudhary,NeerajKumar, andDeepakPandey 1. Introduction 133 2. Basicprinciplesofcentrifugation, 1. Introduction 165 centrifugalforce,andsedimentation 2. Needofproteinpurificationand coefficient 133 determinationofproteinidentity 165 2.1 Calculationofcentrifugalforce 134 3. Basicprinciplesofproteinpurification 166 2.2 Calculationofangularvelocity 134 4. Otherconsiderations 168 2.3 Calculationofrelativecentrifugal 5. Alternativesystems 168 field(RCF) 135 6. Importanceofrecombinantprotein 2.4 Sedimentationcoefficient 135 market 169 3. Instrumentationofacentrifuge 136 7. Conclusion 169 3.1 Typesofrotors 137 References 170 viii Contents 10. Chromatography: Basic principle, 3. IsolationofRNA 198 types, and applications 3.1 Guanidiumthiocyanate-based isolationofRNA 199 MaheshKumarGupta 3.2 TRIzolreagentisolation 199 andPradipKumarBiswas 4. PCRandprimerdesigning 199 1. Basicprinciple 173 4.1 PrimerdesignforPCR 199 2. Generaltermsusedinchromatography 174 4.2 Polymerasechainreaction 200 3. Typesofchromatography 174 4.3 Parametersforprimerpairdesign 200 3.1 Liquidchromatography 174 4.4 Primerdesigntips 201 3.2 Affinitychromatography 176 4.5 Probesdesigntips 201 3.3 Ion-exchangechromatography 177 4.6 Productamplicons 202 3.4 Sizeexclusionchromatography 5. SubmissionofsequencetoGenBank 202 (gelfiltrationchromatography) 180 6. Phylogeneticanalysis 203 3.5 Hydrophobicinteraction 6.1 Methodsofphylogeneticanalysis 203 chromatographyandreversephase 6.2 Character-basedmethods 204 chromatography(hydrophobic 6.3 Calculationofthedegreeof surfacearea) 181 divergence 205 3.6 Multimodalormixed-mode 6.4 Molecularclockhypothesis 205 chromatography(multiple 6.5 Advantagesofphylogeneticanalysis 205 properties) 181 6.6 Modernmethodsinphylogenetic References 182 analysis 205 7. Conclusion 206 11. Electrophoresis: Basic principle, References 206 types, and applications 13. RDTand genetic engineering: BabitaRanaandGopalKrishnaJoshi Basic of RDT method, PCR, and application 1. Introduction 183 2. Typesofelectrophoresis 183 KomaljeetGill,ShivantiNegi,NeerjaRana, 2.1 Gelelectrophoresis 183 andPankajKumar 2.2 Zoneelectrophoresis 189 1. Introduction 207 2.3 Freeflowelectrophoresis 189 2. RecombinantDNAtechnology 208 2.4 Capillaryelectrophoresis 191 3. GeneticengineeringandRDT 3. Conclusion 192 differences 209 References 192 4. Polymerasechainreaction 209 4.1 Denaturation 209 Part III 4.2 Primerannealing 209 4.3 Elongation 210 Genomic and proteomic analysis 4.4 ApplicationsofPCR 210 5. RDT/geneticengineeringlinkageto 12. DNA, RNA isolation, primer bioentrepreneurship 212 designing, sequence submission, 6. Threepillarsofbioentrepreneurship 213 and phylogenetic analysis 7. RecombinantDNAtechnology market-growthandtrends 214 RupaliSharma,ShashwatSharad, 8. RDTlinkedbioentrepreneurshipin GillipsieMinhas,DeepRajSharma, thehealthsector 214 KulsajanBhatia,andNeelKamalSharma 9. RDTlinkedbioentrepreneurshipin 1. Introduction 197 foodandagriculturesector:Helping 2. IsolationofDNA 197 tofeedtheworld 215 2.1 Phenol-chloroformextraction 10. Industrialandenvironmental ofDNA 197 bioentrepreneurship 215 2.2 Detergent-basedisolationofDNA 198 11. Conclusion 215 2.3 Density-gradientcentrifugation 198 References 216 Contents ix 14. Protein sequence analysis 6.10 Fleksy 250 6.11 ParaDockS 251 DeepakSharmaandAbhishekChaudhary 6.12 FLIPDock 251 1. Introduction 217 6.13 PharmDock 251 2. Proteomics 217 6.14 FRED 251 3. Bioinformaticstoolsforprotein 6.15 RosettaLigand 251 sequenceanalysis 218 6.16 FlexibleCDOCKER 251 3.1 DynamicBayesiannetworks 219 6.17 LigandFit 251 3.2 Supportvectormachines 219 6.18 rDock 252 3.3 Neuralnetwork 219 6.19 LeadFinder 252 4. Sequencealigningprograms 220 6.20 GalaxyDock2 252 4.1 Basiclocalalignmentsearchtool 220 6.21 MS-DOCK 252 4.2 FASTA 220 6.22 BetaDock 252 4.3 Clustal 221 6.23 EADock 252 4.4 Otherprograms 221 6.24 FLOG 253 5. Alignment-freesequenceanalysis 221 6.25 Hammerhead 253 6. Conclusion 221 6.26 SwissDock 253 References 222 6.27 DockingServer 253 6.28 1-ClickDocking 253 15. Computationalstrategiesandtools 6.29 DOCKBlaster 253 for protein tertiary structure 6.30 BLINDDockingServer 254 prediction 6.31 ParDOCK 254 6.32 FlexPepDock 254 RajKumarandAjaySharma 6.33 ClusPro 254 6.34 PatchDock 254 1. Introduction 225 6.35 MEDOCK 254 2. Homologymodeling 225 6.36 BSP-SLIM 254 3. Foldrecognition/threading 227 6.37 BioDrugScreen 255 4. Abinitioproteinstructureprediction 236 6.38 KinDOCK 255 5. Hybridmethodsandcurrenttrendin 6.39 idTarget 255 proteinstructureprediction 237 6.40 Pose&Rank 255 6. Conclusion 238 7. Futureprospects 255 References 238 8. Conclusion 256 References 256 16. Docking strategies ArushiSharmaand 17. A beginner’s guide to measuring RagothamanM.Yennamalli binding affinity during 1. Introduction 243 biomolecular interactions 2. Scoringfunctions 245 HannahI.Martin,VidushiAgnihotri, 3. Protein-liganddockingstrategies 246 RagothamanM.Yennamalli,andAurijitSarkar 4. Protein-proteindockingstrategies 247 5. Protein-nucleicaciddockingstrategies 248 1. Introduction 259 6. Differentsoftwareandtoolsusedfor 2. Anintroductiontomajormethodsof docking 249 detectingprotein-ligandcomplexes 261 6.1 AutoDock 249 2.1 Fluorescenceassays 261 6.2 AutoDockVina 249 2.2 Differentialscanningfluorimetry(DSF) 263 6.3 DOCK 249 2.3 Isothermaltitrationcalorimetry 264 6.4 GOLD 249 2.4 Surfaceplasmonresonance 265 6.5 GLIDE 250 2.5 Nuclearmagneticresonance 6.6 GlamDock 250 spectroscopy 266 6.7 FlexAID 250 2.6 Frontalaffinitychromatography 268 6.8 iGEMDOCK 250 3. Conclusion 269 6.9 FlexX 250 References 269