Biotechnology fundamentals / Firdos Alam Khan. – 2nd ed.

– Boca Raton
[etc.], cop. 2016

Spis treści

Preface xxxvii

Author xli

1 Introduction to biotechnology 1
Learning objectives 1
1.1 What is biotechnology? 1
1.1.1 Definitions of biotechnology 2
1.2 Animal biotechnology 4
1.3 Agricultural biotechnology 6
1.4 Medical biotechnology 6
1.5 Industrial biotechnology 7
1.6 Environmental biotechnology 7
1.7 Other emerging fields of biotechnology 8
1.7.1 Nanobiotechnology 8
1.7.2 Bioinformatics 9
1.7.3 Pharmacogenomics 9
1.7.4 Regenerative medicine 9
1.7.5 Therapeutic proteins 10
1.7.6 Biorobotics 11
1.7.7 Biomimetics 11
1.8 History of biotechnology 12
1.8.1 Ancient biotechnology 13
1.8.2 Modern biotechnology 13
1.9 Human Genome Project 14
1.9.1 Need for Human Genome Project 16
1.10 Major scientific discoveries in biotechnology 16
1.11 Biotechnology as the science of integration 18
1.12 Bio-revolution 19
1.13 Ethical and regulatory issues in biotechnology 21
1.14 Future of biotechnology 21
Problems 23
Section A. Descriptive type 23
Section B: Multiple choice 23
Section C: Critical thinking 24
Assignment 24
Online resources 24
References and further reading 25
2 Genes and genomics 27
Learning objectives 27
2.1 Introduction 27
2.2 Cell as the building block of life 28
2.3 Classification of cells 28
2.3.1 Prokaryotic cell 28
2.3.2 Eukaryotic cell 30
2.4 Extracellular organization 30
2.4.1 Cell membrane 30
2.4.2 Cell capsule 31
2.4.3 Flagella 31
2.5 Intracellular organization 32
2 5.1 Cytoplasmic constituents 32
2.5.1.1 Mitochondria and chloroplasts 32
2 5.1.2 Ribosomes 32
2 5.1.3 Endoplasmic reticulum 33
2 5.1.4 Golgi apparatus 33
2 5.1.5 Lysosomes and peroxisomes 33
2 5.1.6 Centrosome 33
2.5.1.7 Vacuoles 33
2.5.1.8 Cytoskeleton 33
2.5.2 Nuclear constituents 34
2.5.2.1 Deoxyribonucleic acid 34
2.5.2.2 Ribonucleic acid 37
2.5.2.3 Messenger ribonucleic acid 38
2 5.2.4 Transfer RNA 38
2 5.2.5 Ribosomal RNA 39
2.5.2.6 Small nuclear RNA 39
2.5.2.7 Nucleolus 40
2.5.2.8 Chromatin 40
2.6 Macromolecules 40
2.6.1 Proteins 40
2.6.2 Carbohydrates 41
2.6.2.1 Simple sugars 42
2.6.2.2 Complex carbohydrates 42
2.6.3 Lipids 43
2.6.4 Epigenetics 43
2.6.4.1 Historical perspective 44
2 6.4.2 Clinical implications 45
2.7 Genes and genetics 47
2.7.1 Mendelian genetics 47
2.7.2 Modern genetics 49
2.8 Cell division 50
2.8.1 Meiosis 50
2.8.2 Mitosis 51
2.9 DNA replication 53
2.9.1 Role of DNA polymerase in replication 53
2 9.2 DNA replication within the cell 55
2.9.2.1 Replication fork 55
2.9.2.2 Leading strand 55
2.9.2.3 Lagging strand 56
2.9.3 Regulation of DNA replication 56
2.9.4 Termination of replication 57
2.10 DNA interactions with proteins 57
2.11 DNA-modifying enzymes 58
2.11.1 Topoisomerases and helicases 59
2.12 DNA methylation 59
2.13 DNA mutation 60
2.14 Tools of biotechnology 61
2.14.1 Polymerase chain reaction 61
2.14.1.1 Principle and practice of PCR 63
2.14.1.2 PCR optimization 65
2.14.2 Types of PCR reactions 65
2.14.2.1 Allele-specific PCR 65
2.14.2.2 Polymerase cycling assembly 65
2.14.2.3 Asymmetric PCR 65
2.14.2.4 Helicase-dependent amplification 66
2.14.2.5 Hot-start PCR 66
2.14.2.6 AFLP-PCR 66
2.14.2.7 Alu PCR 66
2.14.2.8 Colony PCR 67
2.14.2.9 Inverse PCR 67
2.14.2.10 Ligation-mediated PCR 67
2 14.2.11 Methylation-specific PCR 67
2.14.2.12 Miniprimer PCR 67
2.14.2.13 Multiplex ligation- dependent probe amplification 68
2.14.2.14 Multiplex PC 68
2.14.2.15 Nested PCR 68
2.14.2.16 Overlap-extension PCR 68
2.14.2.17 Quantitative PCR 68
2.14.2.18 Reverse transcription PCR 69
2.14.2.19 Solid-phase PCR 69
2.14.2.20 Thermal asymmetric interlaced PCR 69
2.14.2.21 Touchdown PCR (step-down PCR) 69
2.14.2.22 Universal fast walking 69
2.14.3 Applications of PCR 70
2.14.3.1 Diagnostic assay 70
2.14.3.2 Genetic engineering 70
2.14.3.2.1 Forensic DNA profiling 70
Problems 71
Section A: Descriptive type 71
Section B: Multiple choices 71
Section C: Critical thinking 73
Assignment 73
References and further reading 73

3 Proteins and proteomics 75

Learning objectives 75
3.1 Introduction 75
3.2 Significance of proteins 76
3.2.1 Proteins for body functions 77
3.2.1.1 Nonessential amino acids 77
3.2.1.2 Essential amino acids 78
3.2.1.3 Other amino acids 79
3.2.2 Proteins as enzymes 80
3.2.3 Proteins in cell signaling and Ugand binding 80
3.2.4 Structural proteins 81
3.3 Protein biosynthesis 82
3.3.1 Transcription stage 83
3.3.2 Transcription in prokaryotes and eukaryotes 83
3.3.3 Stages of transcription 84
3.3.3.1 Preinitiation 84
3.3.3.2 Initiation 85
3.3.3.3 Promoter clearance 86
3.3.3.4 Elongation 86
3.3.3.5 Termination stage 87
3.3.3.6 Reverse transcription 87
3.3.3.7 RNA splicing 87
3.3.3.8 Spliceosomal introns 88
3.3.3.9 Spliceosome formation and activity 88
3.3.3.10 Self-splicing 88
3.3.3.11 tRNA splicing 88
3.3.3.12 RNA export 88
3.3.4 Translation 89
3.3.4.1 Posttranslational modification 90
3.4 Protein structure 91
3.4.1 Protein prediction methods 93
3.5 Protein folding 93
3.5.1 Tools of protein folding 94
3.5.1.1 Circular dichroism 95
3.5.1.2 Dual polarization interferometry 95
3.5.1.3 Vibration circular dichroism of proteins 95
3.5.1.4 Protein folding with high time resolution 95
3.5.1.5 Energy landscape theory 95
3.6 Protein modification 96
3.6.1 Protein modification by phosphorylation 96
3.6.2 Protein modification by ubiquitination 96
3.6.3 Additional modifications 96
3.7 Protein transport 97
3.8 Protein dysfunction and degradation 97
3.9 Regulation of protein synthesis 101
3.9.1 Stages of gene expression 102
3.9.1.1 Operon model for gene regulation 102
3.9.1.2 General transcription factors 102
3.9.1.3 Enhancers 103
3.9.1.4 Induction and repression 103
3.10 Regulatory protein 105
3.11 Methods for protein analysis 105
3.11.1 Protein identification and quantification 106
3.11.1.1 Native gel 106
3.11.1.2 SDS-PAGE 106
3.11.1.3 QPNC-PAGE 107
3.11.1.4 Protomap 107
3.11.1.5 Northern blot 108
3.11.1.6 Western blot 110
3.11.1.7 Cellular techniques 111
3.11.1.8 Enzyme-linked immunosorbent assay 111
3.12 Protein purification 114
3.13 Tools of proteomics 115
3.13.1 Two-dimensional electrophoresis 115
3.13.2 Mass spectrometry 116
3.13.3 Protein microarray 117
3.13.4 Two-hybrid screening 118
3.13.5 Protein structure prediction 119
3.13.6 PEGylation 119
3.13.7 High-performance liquid chromatography 120
3.13.8 Shotgun proteomics 120
3.13.9 Top-down proteomics 120
Problems 120
Section A: Descriptive type 120
Section B: Multiple choice 121
Section C: Critical thinking 122
Assignment 122
References and further reading 122

4 Recombinant DNA technology 125

Learning objectives 125
4.1 Introduction 125
4.2 Making of recombinant DNA 126
4.2.1 Steps in making a recombinant DNA product 126
4.2.2 Methods involved in making recombinant DNA product 127
4.2.2.1 Transformation 127
4.2.2.2 Nonbacterial transformation 128
4.2.2.3 Phage introduction 128
4.3 Significance of recombinant DNA technology 128
4.4 Role of restriction enzymes in rDNA technology 129
4.4.1 Types of restriction enzymes 129
4.4.1.1 Type I restriction enzymes 129
4.4.1.2 Type II restriction enzymes 130
4.4.1.3 Type III restriction enzymes 131
4.4.2 Nomenclature of restriction enzymes 131
4.4.3 Recognition sequences for type II restriction enzymes 132
4.4.4 Cleavage pattern of type II restriction enzymes 133
4.4.5 Modification of cut ends 133
4.5 Steps in gene cloning 134
4.6 Synthesis of complete gene 134
4.7 Polymerase chain reaction and gene cloning 136
4.7.1 Comparison of PCR versus gene cloning 136
4.7.1.1 How PCR works 136
4.7.1.2 How cloning works 137
4.7.1.3 What are the differences between PCR and cloning? 137
4.8 Significance of vectors in recombinant DNA technology 137
4.8.1 Properties of good vectors 138
4.8.2 Cloning and expression vectors 139
4.8.3 Applications of viral vectors 139
4.9 Classification of vectors 140
4.9.1 Bacterial vectors 141
4.9.1.1 E. coli vectors 141
4.9.1.2 Plasmid vectors 141
4.9.2 Viral vectors 142
4.9.2.1 Retroviruses 142
4.9.2.2 Lentiviruses 144
4.9.2.3 Adenoviruses 145
4.9.2.4 Adeno-associated viruses 145
4.9.3 ARS vectors 146
4.9.4 Minichromosome vectors 146
4.9.5 Yeast artificial chromosome vectors 147
4.9.6 Vectors for animals 148
4.9.7 SV40 vectors 149
4.9.8 Bovine papillomavirus vectors 149
4.10 Integration of the DNA insert into the vector 150
4.10.1 Both ends cohesive and compatible 150
4.10.2 Both ends cohesive and separately matched 151
4.10.3 Both ends cohesive and unmatched 151
4.10.4 Both ends flush/blunt 152
4.10.5 One end cohesive and compatible, the other end blunt 152
4.11 Introduction of the recombinant DNA into the suitable host 152
4.12 Increased competence of E. coli by CaCl2 treatment 152
4.13 Infection by recombinant DNAs packaged as virions 153
4.14 Selection of recombinant clones 153
4.15 Identification of clones having recombinant DNAs 154
4.16 Selection of clone containing a specific DNA insert 155
4.16.1 Colony hybridization 155
4.16.2 Other approaches for developing specific probes 155
4.16.3 Complementation 156
4.16.4 Unique gene products 156
4.16.5 Antibodies specific to the protein product 156
4.16.6 Colony screening with antibodies 157
4.16.7 Fluorescence-activated cell sorter 157
4.17 Applications of recombinant DNA technology 157
4.17.1 Genetically modified organisms 158
4.17.1.1 Transgenic microbes 158
4.17.1.2 Transgenic animals 159
4.17.1.3 Transgenic plants 159
4.17.1.4 Cisgenic plants 160
4.18 DNA sequencing 160
4.18.1 Maxam and Gilbert procedure 160
4.18.2 Enzymatic procedure 162
4.18.3 Automated DNA sequencing 163
4.18.4 Current challenges in DNA sequencing 164
4.18.5 Trends in DNA sequencing 165
4.18.5.1 High-throughput sequencing 165
4.18.5.2 In vitro clonal amplification 165
4.18.5.3 Parallelized sequencing 165
4.18.5.4 Sequencing by ligation 166
4.18.5.5 Microfluidic Sanger sequencing 166
4.18.5.6 Other sequencing technologies 166
4.19 Microarrays 166
4.20 DNA chips 167
4.21 Isolation of desired DNA 168
4.22 cDNA library 169
4.22.1 Problems in cDNA preparation 169
4.22.2 Isolation of mRNA 169
4.23 Preparation of cDNA 170
4.24 Genomic library 170
4.24.1 Construction of a genomic library 170
4.25 DNA libraries 171
4.26 Chemical synthesis gene 171
4.26.1 Phosphodiester approach 172
4.26.2 Phosphotriester approach 172
4.26.3 Phosphite triester approach 173
4.27 Applications of synthetic oligonucleotides 174
Problems 174
Section A: Descriptive type 174
Section B: Multiple choice 174
Section C: Critical thinking 176
Assignments 176
References and further reading 176

5 Microbial biotechnology 179

Learning objectives 179
5.1 Introduction 179
5.2 Structural organization of microbes 180
5.2.1 Structure 180
5.2.2 Intracellular organization 181
5.2.3 Extracellular organization 184
5.3 Microbial metabolism 186
5.3.1 Heterotrophic microbial metabolism 186
5.3.2 Fermentation 188
5.3.3 Aerobic respiration 189
5.3.4 Denitrification 189
5.3.5 Nitrogen fixation 190
5.4 Microbial growth 191
5.4.1 Phases of microbial growth 191
5.4.2 Factors that influence microbial growth 193
5.5 Microbial genetics 193
5.5.1 Mutations 194
5.5.1.1 Auxotropic mutant 194
5.5.1.2 Resistant mutant 195
5.5.1.3 Metabolic mutant 195
5.5.1.4 Regulatory mutant 195
5.5.2 Spontaneous mutations 195
5.5.3 Induction of selective mutations 196
5.5.4 Induced mutations 197
5.6 Genetic recombination in bacteria 197
5.6.1 Bacterial transformation 198
5.6.2 Bacterial transduction 200
5.6.2.1 Types of transduction 201
5.6.2.2 Stages of transduction 204
5.6.3 Conjugation mechanism in gene recombination 204
5.6.4 Human microbiome project 205
5.7 Transposable genetic elements 207
5.7.1 Types of transposable genetic elements 207
5.7.1.1 Insertion sequences 207
5.7.1.2 Transposons 208
5.8 Use of E. coli in microbial cloning 208
5.8.1 Genetic simplicity 208
5.8.2 Growth rate 208
5.8.3 Safety 209
5.8.4 Conjugation and the genome sequence 209
5.8.5 Ability to host foreign DNA 209
5.9 Pathogenic bacteria 209
5.10 Application of microbes 210
5.10.1 Microbes and agriculture 211
5.10.2 Nitrogen fixers 211
5.10.3 Biopesticides and bioweedicides 211
5.10.4 Acetone butanol fermentations 212
5.10.5 Microbes in recovery of metals and petroleum 212
5.10.6 Microbes in the paper industry 212
5.10.7 Microbes in medicine 212
5.10.8 Microbes in synthetic energy fuels 213
5.10.9 Microbes and environment cleaning 213
5.11 Food microbiology 214
5.11.1 Microbes associated with food spoilage 214
5.11.2 Meat and fish 215
5.11.3 Poultry and eggs 215
5.11.4 Breads and bakery products 215
5.11.5 Other foods 215
5.11.6 Importance of microbes in foods 216
5.11.7 Food fermentation 216
5.12 Microbial biotechnology 217
5.12.1 Production of enzymes by microorganisms 218
Problems 218
Section A: Descriptive type 218
Section B: Multiple choice 219
Section C: Critical thinking 220
References and further reading 220

6 Agricultural biotechnology 221

Learning objectives 221
6.1 Introduction 221
6.2 Plant breeding 222
6.2.1 Classical breeding 224
6.2.2 Plant breeding by traditional techniques 224
6.2.2.1 Selection 224
6.2.2.2 Hybridization 224
6.2.2.3 Polyploidy 225
6.2.3 Modern plant breeding 225
6.2.3.1 In vitro cultivation 225
6.2.3.2 In vitro selection and somaclonal variation 226
6.2.3.3 Somatic hybrid plants 227
6.2.3.4 Breeding by restriction fragment length polymorphism 227
6.2.3.5 Plant breeding by gene transfer 229
6.2.3.6 Agrobacterium-mediated gene transfer 229
6.2.3.7 Particle bombardment 230
6.2.3.8 Electroporation and direct DNA entry into protoplasts 230
6.2.3.9 Transgene expression 230
6.2.3.10 Selection and plant regeneration 231
6.2.3.11 Reverse breeding and doubled haploids 233
6.2.3.12 Genetic modification 234
6.3 Plant diseases 234
6.3.1 Diseases caused by fungi 235
6.3.2 Diseases caused by oomycetes 235
6.3.3 Diseases caused by bacteria 236
6.3.4 Diseases caused by plant virus 236
6.3.5 Diseases caused by nematodes 237
6.3.6 Diseases caused by protozoa 237
6.3.7 Diseases caused by parasitic plants 237
6.4 Applications of molecular and genetic tools in agriculture 238
6.4.1 Expression of viral coat protein to resist infection in agriculture 238
6.4.2 Expression of bacterial toxin in agriculture using molecular
techniques 238
6.5 Herbicide-tolerant plants 239
6.6 Pigmentation in transgenic plants 240
6.7 Altering the food content of plants 241
6.8 Gene transfer methods in plants 241
6.9 Target cells for gene transformation 241
6.10 Vectors for gene transfer 242
6.10.1 Structure and functions of Ti and Ri plasmids 242
6.11 Transformation techniques using Agrobacterium 245
6.11.1 Requirements of transgenic plants 245
6.11.2 Explants used for transformation 245
6.11.3 Marker genes for selection and scoring of cells 246
6.11.4 Neomycin phosphotransferase gene 246
6.12 Glucuronidase gene 246
6.13 Agroinfection and gene transfer 247
6.14 DNA-mediated gene transfer 247
6.14.1 Microinjection and macroinjection 248
6.15 Electroporation for gene transfer 248
6.16 Liposome-mediated gene transfer 249
6.17 Gene transformation using pollen 249
6.18 Application of transgenic plants 250
6.18.1 Detoxification or degradation of herbicides 250
6.18.2 Crystal (cry) proteins 250
6.18.3 Toxic action of cry proteins 251
6.18.4 Expression of cry genes in plants 251
6.18.5 Insect resistance to cry proteins 251
6.18.6 Virus resistance 252
6.18.7 Drought resistance 252
6.18.8 Modification of seed protein quality 253
6.18.8.1 Introduction of an appropriate transgene 253
6.18.8.2 Modification of endogenous genes 254
6.18.8.3 Successful examples 254
6.18.9 Cosuppression of genes 254
6.18.10 RNA-mediated interference 255
6.18.11 Biochemical production in plants 256
6.18.12 Plant-derived vaccines 256
6.18.12.1 Edible vaccines 256
6.18.12.2 Recombinant and subunit vaccines 257
6.18.13 Hirudin: A polypeptide 258
6.18.14 Phytase as an enzyme 258
6.18.15 Polyhydroxybutyrate biodegradable plastic substrate 258
6.19 How safe are transgenic plants? 259
6.20 Bioengineered plants 259
6.20.1 Golden rice 260
6.20.2 Tomato "Flavr Savr" 262
6.21 Genetically modified maize 263
6.22 Terminator technology 265
6.22.1 Types of terminator technology 265
6.22.1.1 V-GURT 265
6.22.1.2 T-GURT 266
6.22.2 Benefits of terminator technology 266
6.22.3 Concerns of terminator technology 266
Problems 267
Section A: Descriptive type 267
Section B: Multiple choice 267
Section C: Critical thinking 268
Assignment 268
References and further reading 269

7 Animal biotechnology 271

Learning objectives 271
7.1 Introduction 271
7.2 History of the use of animals in research 273
7.3 Drug testing in animals is mandatory 274
7.4 Most commonly used animals in research 274
7.4.1 Invertebrates 275
7.4.2 Fish and amphibians 276
7 4.3 Vertebrates 276
7.4.3.1 Rodents 276
7.4.3.2 Cats and dogs 276
7.4.3.3 Primates and nonprimates 277
7.5 Application of animal models 277
7.5.1 Use of animals in basic research 218
7.5.2 Use of animals in applied research 279
7.5.2.1 Genetic diseases 279
7.5.2.2 Virology 279
7.5.2.3 Neurological disorders 279
7.5.2.4 Organ transplantation 280
7.5.2 5 Drug efficacy testing 280
7.5.2.6 Toxiсоlogical analysis 281
7.5.2.7 Cosmetics testing 283
7.6 Animal models 284
7.6.1 Caenorhabditis elegans 284
7.6.2 Drosophila melanogaster 286
7.6.3 Laboratory mouse 287
7.6.4 Rhesus monkey 287
7.6.5 Xenopus laevis 288
7.6.6 Zebrafish 289
7.6.7 Bioengineered mosquito 290
7.7 Animal biotechnology 291
7.7.1 Use of animals in antibody production 291
7.7.1.1 Monoclonal antibodies in diagnostic applications 291
7.7.1.2 Monoclonal antibodies in therapeutic applications 291
7.7 1.3 Recombinant antibodies 293
7.7.2 In vitro fertilization and embryo transfer 293
7.7.2.1 Embryo transfer in cattle 293
7.7.3 Animal cell culture products 295
7.7.4 Animal cloning 295
7.7.4.1 Cloning of extinct and endangered species 296
7.7.5 Transgenic animals 297
7.7.5.1 Transgenic cow 300
7.7.5.2 Transgenic pigs 300
7.7.5.3 Transgenic goat 301
7.7.5.4 Transgenic sheep 301
7.7.5.5 Transgenic chickens 302
7.7.5.6 Transgenic primates 303
7.8 Biotechnology and fish farming 303
7.8.1 Mariculture 303
7.8.2 Polyculture 304
7.8.3 Aquatic biotechnology 304
7.8.3.1 Transgenic fish 304
7.8.3.2 Green fluorescent protein 305
7.8.3.3 Antifreeze proteins 305
7.8.3.4 Transgenic salmon 306
7.8.3.5 Transgenic mussel 307
7.8.3.6 Fugu fish 307
7.8.3.7 Squalus acanthias 308
7.8.3.8 Limulus polyphemus 308
7 9 Regulations of animal testing 309
7.10 Alternatives to animal testing 310
7.10.1 n vitro cell culture technique 310
7.10.2 Synthetic membranes 311
7.10.3 Statistics instead of animal testing 311
7.10.4 Newer scanning techniques 311
7.10.5 Computer models 312
Problems 312
Section A: Descriptive type 312
Section B: Multiple choices 313
Section C: Critical thinking 314
Assignment 314
References and further reading 314

8 Environmental biotechnology 317

Learning objectives 317
8.1 Introduction 317
8.1.1 Ecosystem 317
8.1.2 Biomes 318
8.1.3 Wilderness 318
8.1.4 Geological activity 319
8.1.5 Oceanic activity 319
8.2 Factors affecting the environment 320
8.2.1 Global warming 320
8.2.1.1 Carbon footprint 320
8.2.1.2 Destruction of forests 320
8.2.1.3 Air pollution 322
8.2.1.4 Major carbon dioxide emission countries 324
8.2.1.5 Greenhouse effect 324
8.2.1.6 Acid rain 324
8.2.1.7 Ocean acidification 325
8.2.1.8 Health hazards due to pollution 325
8.3 Environment protection by biotechnology 326
8.3.1 Bioremediation 327
8.3.1.1 Mycoremediation 329
8.3.2 Wastewater treatment 329
8.3.2.1 Pretreatment phase 330
8.3.2.2 Screening phase 330
8.3.2.3 Sedimentation phase 331
8.3.2.4 Secondary treatment phase 331
8.3.2.5 Activated sludge 332
8.3.2.6 Filter beds 332
8.3.2.7 Biological aerated filters 332
8.3.2.8 Nutrient removal 332
8.3.2.9 Nitrogen removal 333
8.3.2.10 Phosphorus removal 333
8.3.2.11 Disinfection of wastewater 334
8.3.2.12 Sludge disposal 334
8.3.3 Biofuels 335
8.3.3.1 Biodiesel 337
8.3.3.2 Biogas 338
8.3.3.3 Bioalcohols 338
8.3.3.4 Bioethers 339
8.3.3.5 Syngas 339
8.3.3.6 Solid biofuels 339
8.3.3.7 Second-generation biofuels 340
8.3.3.8 Third-generation biofuels 340
8.3.3.9 International biofuel efforts 340
8.3.3.10 Future of biofuels 340
8.3.4 Biodegradable plastic 341
8.3.5 Biodegradation by bacteria 342
8.3.6 Oil-eating bacteria 343
8.3.7 Biostimulation and bioaugmentation 345
8.3.8 Вioleaching 346
8.3.9 Single-cell protein and biomass from waste 347
8.3.10 Vermitechnology 347
8.3.11 Biosorption 348
8.3.11.1 Bacteria 348
8.3.11.2 Fungi 349
8.3.11.3 Algae 350
8.3.12 Genetically engineered organisms 350
Problems 350
Section A: Descriptive type 350
Section В: Multiple choice 350
Section C: Critical thinking 352
Debate 352
Field visit 352
References and further reading 352

9 Medical biotechnology 355

Learning objectives 355
9.1 Introduction 355
9.1.1 Vaccine development 355
9.1.1.1 Killed vaccines 356
9.1.1.2 Attenuated vaccines 356
9.1.1.3 Toxoid vaccines 357
9.1.1.4 Subunit vaccines 357
9.1.1.5 Conjugate vaccines 357
9.1.1.6 Experimental vaccines 358
9.1.1.7 Valence vaccines 358
9.1.1.8 Vaccine production 358
9.1.1.9 Making of influenza vaccines 360
9.1.1.10 Large-scale production of vaccines 362
9.1.1.11 Economics involved in vaccine production 364
9.1.1.12 Trends in vaccine research 364
9.1.1.13 Issues related to vaccines 366
9.1.1.14 Synthetic peptides as vaccines 366
9.2 Antibody production 367
9.2.1 Applications of monoclonal antibodies 368
9.2.1.1 Diagnostic test 369
9.2.1.2 Cancer treatment 369
9.2.2 Hybridoma technology 369
9.2.2.1 Purification of monoclonal antibodies 370
9.2.3 Recombinant monoclonal antibodies 372
9.2.4 Constraints in making monoclonal antibodies 372
9.3 Therapeutic proteins 373
9.3.1 Growth factors 374
9.4 Stem cell transplantation 375
9.4.1 Adult stem cells 375
9.4.1.1 Dental pulp-derived stem cells 377
9.4.1.2 Hematopoietic stem cells 377
9.4.1.3 Mammary stem cells 377
9.4.1.4 Mesenchymal stem cells 378
9.4.1.5 Neural stem cells 378
9.4.1.6 Olfactory adult stem cells 379
9.4.1.7 Clinical applications of stem cells 379
9.4.1.8 Cancer treatment 380
9.4.1.9 Spinal cord injury 381
9.4.1.10 Corneal repair 381
9.4.2 Embryonic stem cells 382
9.4.2.1 How were embryonic stem cells discovered? 382
9.4.2.2 Cell line contamination 382
9.4.2.3 Immunorejection 384
9.4.2.4 Alternative approach to creating embryonic stem cells 384
9.4.2.5 Embryonic stem cells as models for human genetic disorders 385
9.4.2.6 First clinical trial 385
9.5 Bioengineered skin 386
9.6 Bioengineered organ transplantation 387
9.7 Gene therapy 387
9.7.1 Ex vivo gene therapy 388
9.7.1.1 Gene therapy using viral vectors 389
9.7 1.2 Nonviral methods of DNA transfer 393
9.7.2 In vivo gene therapy 396
9.7.3 Problems with gene therapy 397
9.7.3.1 Short-lived nature of gene therapy 397
9.7.3.2 Immune response 397
9.7.3.3 Virus-induced toxicity 397
9.7.3.4 Not for multigene disorders 397
9.7.3.5 Induced mutagenesis 398
9.7.4 Genetic counseling 398
9.8 Molecular diagnostics 399
9.8.1 DNA fingerprinting 399
9.8.2 Techniques of DNA profiling 401
9.8.2.1 Restriction fragment length polymorphism analysis 401
9.8.2.2 PCR analysis 402
9.8.2.3 Short tandem repeat analysis 402
9.8.2.4 Amplified fragment length polymorphism analysis 403
9.8.2.5 Y chromosome analysis 403
9.8.2.6 Mitochondrial DNA analysis 404
9.8.2.7 Confirmation of genetic relationship 404
9.8.2.8 Fake DNA evidence 404
9.8.2.9 Criminal DNA data 405
9.9 Artificial blood 405
9.10 Organ transplant 407
9.10.1 History of organ transplant 407
9.10.2 Types of transplants 409
9.10.2.1 Autograft 409
9.10.2.2 Allograft 410
9.10.2.3 Isograft 410
9.10.2.4 Xenograft 411
9.11 Cloning 411
9.11.1 Molecular cloning 411
9.11.2 Clonal cell technology 412
9.11.3 Clonal embryo 413
9.11.4 Reproductive cloning 413
9.11.5 Human Cloning 414
Questions 415
Section A: Descriptive type 415
Section B: Multiple choice 415
Section C: Critical thinking 417
Assignment 417
References and further reading 417

10 Nanobiotechnology 421
Learning objectives 421
10.1 Introduction 421
10.2 Nanotechnology 421
10.3 Nanobiotechnology 423
10.4 Applications of nanobiotechnology 424
10.4.1 Nanomedicine 424
10.4.1.1 Drug delivery 425
10.4.1.2 Cancer diagnostics 428
10.4.1.3 In vivo drug imaging 431
10.4.1.4 Nanonephrology 432
10.4.1.5 Gene therapy using nanotechnology 433
10.4.1.6 Antimicrobial techniques 434
10.4.2 Neuro-electronic interfaces 434
10.4.3 Molecular nanotechnology 435
10.4.4 Nanorobots 435
10.4.5 Cell repair machines 435
10.4.6 Nanosensors 436
10.4.7 Nanoparticles 438
10.4.7.1 Classification of nanoparticles 439
10.4.7.2 Characterization of nanoparticles 439
10.4.7.3 Nanoparticles and safety issues 439
10.5 Nanotechnology in the food industry 440
10.6 Water pollution and nanotechnology 441
10.7 Research trends 441
Problems 442
Section A: Descriptive type 442
Section B: Multiple choice 442
Section C: Critical thinking 443
References and further reading 443

11 Product development in biotechnology 445

Learning objectives 445
11.1 Introduction 445
11.2 Methods of scientific inquiry 446
11.2.1 Characterizations of scientific investigation 447
11.2.2 Scientific inventions 448
11.3 Commercialization of scientific discovery 449
11.4 Business plan 449
11.4.1 Project feasibility 450
11.4.1.1 Market research 450
11.4.1.2 Significance of a project 450
11.4.1.3 Technical outline 450
11.4.1.4 Time plan 450
11.4.1.5 Project cost 450
11.4.1.6 Legal and regulatory issues 451
11.4.1.7 Quality of the product 451
11.5 Biotechnology product development 451
11.5.1 Infrastructure requirements 451
11.5.1.1 Research and development facility 453
11.5.1.2 Animal testing facility or preclinical lab 454
11.5.1.3 Bioequivalence lab 456
11.5.1.4 Clinical trial center 456
11.5.1.5 Manufacturing plant 457
11.5.1.6 Formulation lab 457
11.5.1.7 Quality assurance and quality control lab 458
11.6 Phases of biotechnology product development 458
11.6.1 Preclinical studies 459
11.6.2 Phase 0 459
11.6.3 Phase I 460
11.6.3.1 Single ascending dose 460
11.6.3.2 Multiple ascending doses 461
11.6.3.3 Food effect 461
11.6.4 Phase II 461
11.6.5 Phase III 461
11.6.6 Phase IV 462
11.7 Biotechnology entrepreneurship 463
11.7.1 Starting a biotechnology company 463
11.7.1.1 Grants 463
11.7.1.2 Private investors 464
11.7.1.3 Angel investors 464
11.7.1.4 Venture capitalists 464
11.7.1.5 Bank loans 464
11.8 Biotechnology industry: Facts and figures 464
11.8.1 Capital investment in biotechnology 466
11.8.2 Mergers and acquisitions of biotechnology companies 467
11.9 Formation of a new biotechnology company 467
11.10 Successful bioentrepreneurship 468
11.11 Biotechnology products and intellectual property rights 468
11.11.1 Patenting, licensing, and partnership in the biotechnology
industry 469
11.11.2 Intellectual property protection 470
11.11.3 Intellectual property rights for plants 471
11.11. 4 Patents and biotechnology products 472
11.11.5 International treaties 473
11.11.5.1 Patent Cooperation Treaty 474
11.11.5.2 World Intellectual Property Organization 475
11.11.5.3 Agreement on Trade-Related Aspects of Intellectual
Property Rights 475
11.11.5.4 Issues with biotechnology patents 476
11.11.5.5 Patent infringements 477
11.12 Biotechnology stock investment: Pros and cons 478
11.12.1 Products in the pipeline 479
11.12.2 Collaboration and merger 479
11.12.3 Experienced management 479
11.12.4 Cash flow 480
11.13 Marketing trends in biotechnology 480
11.14 Role of regulators in biotechnology product development 480
11.14.1 World Health Organization 480
11.14.2 International Conference on Harmonization 481
11.14.3 United States Food and Drug Administration 482
11.14.4 Good laboratory practice 484
11.14.4.1 GLP requirements 484
11.14.4.2 National legislation 484
11.14.4.3 Facilities 485
11.14.4.4 GLP inspection and enforcement 486
11.14.5 Good manufacturing practice 486
11.14.5.1 Cleanroom facility 487
11.14.5.2 Cleanroom classifications 487
11.14.5.3 GMP enforcement 487
11.15 Certification and accreditation 488
11.15.1 International Organization for Standardization 488
11.15.1.1 ISO 14644-1 489
11.15.1.2 ISO 14698-1 489
11.15.1.3 ISO 14698-2 489
11.15.2 British Standard 5295 489
Problems 490
Section A: Descriptive type 490
Section B: Multiple choice 490
Section C: Critical thinking 491
Field trip 491
References and further reading 491

12 Industrial biotechnology 493

Learning objectives 493
12.1 Introduction 493
12.2 Fermenter or bioreactor 494
12.3 Principle of fermentation 495
12.3.1 Aerobic fermentation 496
12.3.1.1 Submerged culture method 496
12.3.1.2 Semisolid/solid-state methods 496
12.3.2 Anaerobic fermentation 497
12.3.2.1 Batch fermentation process 498
12.3.2.2 Continuous fermentation process 499
12 4 Production of biomolecules using fermenter technology 499
12.4.1 Gluconic acid 499
12.4.2 Citric acid 499
12.4.3 Acetone butanol 500
12.4.4 Itaconic acid 500
12.4.5 Gibberellic acid 500
12.4.6 Lactic acid 501
12.4.7 Amino acids 501
12.4.8 Enzymes 502
12.4.9 Proteases 502
12.4.10 Amylases 503
12.5 Development process of microbial products 503
12.5.1 Isolation and screening of microorganisms 503
12.5.1.1 Isolation of microorganisms 504
12.5.1.2 Screening of microorganisms 505
12.5.2 Inoculum development 506
12.5.3 Culture media 506
12.5.4 Contamination 506
12.5.5 Sterilization 507
12.5.5.1 Heating 507
12.5.5.2 Radiation 507
12.5.5.3 Chemicals 508
12.5.5.4 Filtration 508
12.5.6 Strain improvement 508
12.5.6.1 Mutant selection 508
12.5.6.2 Selective isolation of mutants 509
12.6 Upstream bioprocess 510
12.6.1 Industrial microbial culture 510
12.6.2 Mammalian cell culture 512
12.6.2.1 Manipulation of cultured cells 512
12.6.2.2 Generation of hybridomas 513
12.6.3 Nonmammalian culture methods 513
12.6.3.1 Industrial plant cell culture 513
12.6.3.2 Bacterial/yeast culture methods 514
12.6.3.3 Viral culture methods 514
12.7 Downstream bioprocess 514
12.7.1 Stages in DSP 514
12.7.1.1 Removal of insolubles 515
12.7.1.2 Product isolation 515
12.7.1.3 Product purification 515
12.7.1.4 Product polishing 515
12.8 Bioprocess automation 515
12.8.1 Modeling individual unit operations 516
12.8.2 Simple mass balance 517
12.8.3 Mass and heat balance 517
12.8.4 Rate-based model 517
12.8.5 Batch processing 517
12.8.6 Continuous processes 518
12.8.7 Dynamic Simulation 518
12.8.8 Water consumption 518
12.8.9 Waste recycling 518
12.9 Industrial application of microbes 519
12.9.1 Corynebacterium 519
12.9.2 Bacillus 519
12.9.3 Saccharomyces cerevisiae 520
12.9.4 Pseudomonas 520
12.9.5 Clostridium 521
12.9.6 Thermophiles 521
12.10 Industrial production of healthcare products 522
12.10.1 Antibiotic manufacturing 522
12.10.1.1 Penicillin production 522
12.10.1.2 Cephalosporins production 523
12.10.1.3 Streptomycin production 523
12.10.2 Steroids production 526
12.10.3 Textile production 526
12.10.4 Microbial synthesis of vitamin B12 527
Problems 527
Section A: Descriptive type 527
Section B: Multiple choice 528
Section C: Critical thinking 529
Laboratory assignment 529
References and further reading 529

13 Ethics in biotechnology 531

Learning objectives 531
13.1 Introduction 531
13.2 Genetically modified foods and plants 531
13.3 Use of animals as experimental models 534
13.3.1 Disadvantages of animal testing 536
13.3.2 Attacks on researchers 536
13.3.3 Regulations for animal testing in the United States 537
13.3.4 Role of animal welfare groups 537
13.3.5 Future of animal testing 538
13.3.6 Decide for yourself 538
13.4 Use of humans as experimental models 539
13.4.1 Bioethical implications 540
13.5 Xenotransplantation 541
13.6 Genetic screening 543
13.7 Biometrics 544
13.8 DNA fingerprinting 545
13.9 Organ donation and transplantation 545
13.10 Euthanasia 546
13.11 Neuroethics 547
13.12 Assisted reproductive technology 547
13.13 Embryonic stem cell research 548
13.13.1 In favor of embryonic stem cell research 550
13.13.2 Against embryonic stem cell research 550
13.13.3 Current status 551
13.14 Human cloning 551
Questions 553
Section A: Descriptive type 553
Section B: Multiple choice 553
Section С: Critical thinking 553
Assignments 554
References and further reading 555
Careers in biotechnology 557

14 Learning objectives 557

14.1 Introduction 557
14.2 Education and investment in biotechnology 557
14.3 Research and development in biotechnology 561
14.4 Biotechnology industry and products 563
14.5 Biotechnology status in the United States 563
14.6 Career opportunities in biotechnology 564
14.7 Entry-level job positions in biotechnology 566
14.7.1 Research and development division 566
14.7.1.1 Glass washer 566
14.7.1.2 Laboratory assistant 566
14.7.1.3 Research associate 567
14.7.1.4 Research assistant 567
14.7.1.5 Postdoctoral fellow 567
14.7.1.6 Media preparation technician 567
14.7.1.7 Greenhouse assistant 567
14.7.1.8 Plant breeder 567
14.7.2 Quality control 568
14.7.2.1 Quality control analyst 568
14.7.2.2 Quality control engineer 568
14.7.2.3 Environmental health and safety specialist 568
14.7.2.4 Quality assurance auditor 569
14.7.2.5 Validation engineer 569
14.7.2.6 Validation technician 569
14.7.3 Clinical research 569
14.7.3.1 Clinical research administrator 569
14.7.3.2 Clinical coordinator 570
14.7.3.3 Clinical programmer 570
14.7.3.4 Biostatistician 570
14.7.3.5 Clinical data specialist 570
14.7.3.6 Drug experience coordinator 571
14.7.3.7 Clinical research associate 571
14.7.3.8 Animal handler 571
14.7.3.9 Animal technician 571
14.7.3.10 Technical writer 571
14.7.4 Product and development 572
14.7.4.1 Product development engineer 572
14.7.4.2 Production planner/scheduler 572
14.7.4.3 Manufacturing technician 572
14.7.4.4 Packaging operator 573
14.7.4.5 Manufacturing research associate 573
14.7.4.6 Instrument/ calibration technician 573
14.7.4.7 Biochemical development engineer 573
14.7.4.8 Process development associate 574
14.7.4.9 Assay analyst 574
14.7.4.10 Manufacturing engineer 574
14.7.5 Regulatory affairs 574
14.7.5.1 Regulatory affairs specialists 574
14.7.5.2 Documentation coordinator 575
14.7.5.3 Documentation specialist 575
14.7.6 Information systems 575
14.7.6.1 Library assistant 575
14.7.6.2 Scientific programmer/analyst 575
14.7.7 Marketing and sales 576
14.7.7.1 Market research analyst 576
14.7.7.2 Systems analyst 576
14.7.7.3 Sales representative 576
14.7.7.4 Customer service representative 576
14.7.7.5 Technical service representative 577
14.8 Administration 577
14.8.1 Technical recruiter 577
14.8.2 Human resource representative 577
14.8.3 Patent administrator 577
14.8.4 Patent agent 578
14.9 Which job is good for me? 578
14.10 Why are R&D jobs the most challenging? 580
14.10.1 Medical and diagnostics sectors 580
14.10.1.1 Detecting and treating hereditary diseases 580
14.10.1.2 Heart disease 580
14.10.1.3 Cancer 580
14.10.1.4 AIDS 580
14.10.1.5 Veterinary medicine 581
14.10.1.6 Vaccines 581
14.10.1.7 Monoclonal antibodies 581
14.10.1.8 Growth hormones 581
14.10.2 Agricultural sector 581
14.10.2.1 Crop yield 581
14.10.2.2 Protein and oil content of seeds 582
14.10.2.3 Environmental conditions 582
14.10.2.4 Disease and pest resistance 582
14.10.3 Law enforcement sector 582
14.10.4 Product manufacturing sector 582
14.10.5 Microbial engineering sector 583
14.11 Salary and incentives in biotechnology 583
Problems 584
Assignments 585
Field visit 585
References and further reading 585

15 Laboratory tutorials 587

15.1 Laboratory experiments 587
15.1.1 Controlled experiment 588
15.1.2 Observational experiment 589
15.2 Laboratory safety 589
15.3 Good laboratory practices for biotechnology labs 591
15.4 General laboratory techniques 593
15.4.1 Pipetting technique 593
15.4.1.1 Plastic pipette 593
15.4.1.2 Plastic pipette pump 593
15.4.1.3 Pipette with a bulb 594
15.4.1.4 Micropipette 594
15.4.2 Centrifugation technique 595
15.4.3 Spectrophotometer technique 596
15.4.4 Aseptic techniques 597
15.5 General principles of animal handling 598
15.6 Animal anesthesia 599
15.6.1 Anesthesia by gas 599
15.6.2 Injectable anesthesia 600
15.6.2.1 Intravenous method 600
15.6.2.2 Intramuscular method 600
15.6.2.3 Intraperitoneal method 601
15.6.2.4 Subcutaneous method 601
15.6.3 Animal euthanasia 601
15.6.3.1 Criteria for euthanasia 601
15.6.3.2 Surgical operation 602
15.6.3.3 Use of a CO2 chamber 602
15.7 Animal histology 602
15.8 Blood collection in animals 605
15.9 Histology and microscopy 607
15.9.1 Immunocytochemistry technique 607
15.9.1.1 Direct immuno-fluorescence 607
15.9.1.2 Indirect immuno-fluorescence 609
15.10 Separation techniques 610
15.10.1 Agarose gel electrophoresis (basic method) 610
15.10.2 How much percentage gel will be made? 610
15.10.3 Which gel tank to use? 611
15.10.4 How much DNA should be loaded? 611
15.10.5 Which comb? 611
15.11 Microbiology techniques 614
15.11.1 Isolation of pure culture 614
15.11.2 Streaking bacteria for single colonies 616
15.11.3 Gram-staining procedure 616
15.12 Biochemistry techniques 617
15.12.1 Estimation of fat in milk samples 617
15.12.2 Protein quantification by Bradford assay 618
15.12.3 Indirect ELISA 619
15.12.4 Sandwich ELISA 621
15.12.5 Sonication of bacteria 621
15.13 Molecular techniques 622
15.13.1 Isolation of genomic DNA from blood 622
15.13.2 Isolation of DNA from fresh or frozen tissue 623
15.13.3 Preparation of genomic DNA from bacteria 626
15.13.4 DNA isolation procedure 626
15.13.5 Polymerase chain reaction 627
15.13.6 Semiquantitative RT-PCR 630
15.13.6.1 Role of housekeeping gene transcript 630
15.13.6.2 Isolation of total RNA 631
15.14 Genetic techniques 633
15.14.1 Preparation of human metaphase chromosomes 633
15.14.2 Structural analysis of human chromosomes by karyotype 635
15.14.3 DNA amplification fingerprinting protocol 637
15.14.4 Single-strand conformation polymorphism technique 638
15.15 Agricultural biotechnology 640
15.15.1 Plant DNA isolation 640
15.15.2 Plant regeneration by protoplast fusion 641
15.15.3 Simplified Arabidopsis transformation 643
15.15.4 Agrobacterium-mediated gene transfer via hypocotyls 644
15.15.5 Isolation of DNA from onion 644
15.15.6 Isolation of DNA from wheat germ 645
15.16 Microbial biotechnology 646
15.16.1 Gram-positive/Gram-negative staining 646
References 647

Index 649

oprac. BPK