1.The Development of Molecular Biotechnology 1
Emergence of Molecular Biotechnology 1
Recombinant DNA Technology 3
Commercialization of Molecular Biotechnology 6
Concerns and Consequences 8
Summary 10
References 10
Review Questions 11
2.Fundamental Technologies 13
Molecular Cloning 13
Preparation of DNA for Cloning 13
Insertion of Target DNA into a Plasmid Vector 18
Transformation and Selection of Cloned DNA in a Bacterial Host 21
Cloning Eukaryotic Genes 26
Recombinational Cloning 30
Genomic Libraries 32
Genome Engineering Using CRISPR Technology 36
Polymerase Chain Reaction 39
Amplification of DNA by PCR 39
Cloning PCR Products 42
Quantitative PCR 44
Chemical Synthesis of Genes 46
Assembling Oligonucleotides into Genes 46
Assembling PCR Products into Genes 46
DNA Sequencing Technologies 48
Dideoxynucleotide Sequencing 50
Sequencing Using Reversible Chain Terminators 53
SingleMolecule RealTime Sequencing 55
Nanopore Sequencing 56
Sequencing Whole Genomes 56
Preparation of Genomic DNA Sequencing Libraries 57
HighThroughput NextGeneration Sequencing 59
Genome Sequence Assembly 60
Sequencing Metagenomes 61
Genomics 62
Transcriptomics 65
Proteomics 70
Metabolomics 84
Summary 86
References 87
Review Questions 89
3.Production of Recombinant Proteins 91
Protein Production in Prokaryotic Hosts 91
Regulating Transcription 92
Increasing Translation Efficiency 96
Increasing Protein Stability 100
Increasing Protein Secretion 105
Facilitating Protein Purification 111
Integrating DNA into the Host Chromosome 114
Heterologous Protein Production in Eukaryotic Cells 119
Posttranslational Modification of Eukaryotic Proteins 120
General Features of Eukaryotic Expression Systems 123
Yeast Expression Systems 123
BaculovirusInsect Cell Expression Systems 137
Mammalian Cell Expression Systems 145
Protein Engineering 156
Directed Mutagenesis 156
Random Mutagenesis 161
Examples of Protein Engineering 164
Summary 173
References 174
Review Questions 176
4.Molecular Diagnostics 179
Immunological Approaches To Detect Protein Biomarkers 180
Antibodies 180
Agglutination 185
EnzymeLinked Immunosorbent Assays 186
Protein Arrays To Detect Polygenic Diseases 194
Immunoassays for Protein ConformationSpecific Disorders 197
DNABased Diagnostic Approaches 199
Hybridization Probes 199
PCRBased Detection Methods 207
CRISPRCasBased Diagnostic Assays 218
DNA Microarrays 219
WholeGenome Sequencing To Assess Genetic Disease Risk 225
Detecting RNA Signatures of Disease 226
Detection of DiseaseAssociated Changes in Gene Expression 227
Detection of RNA Signatures of Antibiotic Resistance in Bacteria 228
Detection of miRNA Signatures of Disease 230
Biofluorescent and Bioluminescent Systems 233
Fluorescent Proteins 233
Luciferase 234
Microbial Biosensors 235
Summary 238
References 239
Review Questions 241
5.Protein Therapeutics 243
Pharmaceuticals 244
Human Interferons 244
Human Growth Hormone 248
Tumor Necrosis Factor Alpha 251
Extending Protein Half-Life 252
Enzymes 253
DNase I 253
Alginate Lyase 254
Phenylalanine Ammonia Lyase 258
1 -Antitrypsin 259
Glycosidases 261
Masking Nonhuman Epitopes 263
Toxin-Intein Fusions 264
Targeting Mitochondria 265
Bacteria and Therapeutics 267
Interleukin-10 270
Leptin 272
An HIV Inhibitor 274
Insulin 276
Parkinsons Disease 279
Cancer and Bacteria 279
Recombinant Antibodies 280
Hybrid HumanMouse Monoclonal Antibodies 284
Human Monoclonal Antibodies 287
Antibody Fragments 289
Combinatorial Libraries of Antibody Fragments 294
A Combinatorial Library of Full-Length Antibodies 297
Shuffling CDR Sequences 298
Dual-Variable-Domain Antibodies 298
Bispecific Antibodies against Hemophilia 300
Anti-HIV Antibodies 300
Anticancer Antibodies 302
Antibodies against Various Diseases 309
Antiobesity Antibodies 313
Enhanced Antibody Half-Life 315
Affibody Molecules 315
Summary 318
References 318
Review Questions 322
6. Nucleic Acids as Therapeutic Agents 325
Targeting Specific mRNA and DNA Sequences 327
Antisense RNA 327
Aptamers 331
Ribozymes and DNAzymes 338
Interfering RNA 341
Zinc Finger Nucleases 348
CRISPRCas System 349
Nanozymes 351
Nanoparticles 352
Engineering Bacteriophages 352
Viral Delivery Systems 357
Nonviral Delivery Systems 365
Direct Injection 365
Lipids 367
Bacteria 369
Dendrimers 372
Antibodies 373
Aptamers 373
Transposons 374
Gene Therapy 376
Mitochondrial Diseases 378
Prodrug Activation Therapy 378
Promoterless Gene Targeting 379
Summary 382
References 382
Review Questions 386
7. Vaccines 387
Vaccination 387
Current and Future Vaccines 389
Subunit and Peptide Vaccines 392
Herpes Simplex Virus 393
Bovine Herpes Virus-1 394
Cholera 396
Influenza 396
SARS 397
COVID-19 399
Staphylococcus aureus 401
Human Papillomavirus 402
Foot-and-Mouth Virus 404
Streptococcus 405
Peptides 407
Malaria 408
Delivery 411
Genetic Immunization: DNA Vaccines 414
Delivery 414
Cancer 422
Zika Virus 422
Dental Caries 423
Engineered Attenuated Vaccines 424
Herpes Simplex Virus 425
Cholera 426
Salmonella Species 428
Leishmania Species 430
Vector Vaccines 430
Vaccines Directed against Viruses 430
Vaccines Directed against Bacteria 441
Bacteria as Antigen Delivery Systems 444
Monoclonal Antibody Passive Immunity 449
Influenza Virus 450
Summary 452
References 452
Review Questions 456
8. Industrial and Environmental Uses of Recombinant Microorganisms 459
Restriction Endonucleases 459
Small Biological Molecules 461
l-Ascorbic Acid 463
Indigo 467
Amino Acids 468
Lycopene 473
Antibiotics 474
Biopolymers 487
Solvent Tolerance 493
Systems Metabolic Engineering To Optimize Product Yield 494
Microbial Degradation of Xenobiotics 496
Genetic Engineering of Biodegradative Pathways 497
Plastics 507
Utilization of Starch and Sugars 508
Commercial Production of Fructose and Alcohol 508
Increasing Alcohol Production 510
Improving Fructose Production 517
Utilization of Cellulose and Hemicellulose 518
Lignocellulosics 519
Cellulase Genes 522
Direct Conversion of Biomass to Ethanol 530
Alcohol Production by Zymomonas mobilis 531
Lipids from Cyanobacteria 534
Hydrogen Production 535
Summary 538
References 539
Review Questions 542
9. Large-Scale Production of Proteins and Nucleic Acids from Recombinant Microorganisms 545
Principles of Microbial Growth 547
Batch Fermentation 548
Fed-Batch Fermentation 549
Continuous Fermentation 550
Maximizing the Efficiency of the Fermentation Process 551
High-Density Cell Cultures 552
Increasing Plasmid Stability 555
Quiescent E. coli Cells 555
Protein Secretion 558
Reducing Acetate 558
Improving Antibody Production in E. coli 561
Bioreactors 561
Typical Large-Scale Fermentation Systems 565
Two-Stage Fermentation in Tandem Airlift Reactors 566
Two-Stage Fermentation in a Single Stirred-Tank Reactor 568
Batch versus Fed-Batch Fermentation 569
Harvesting Microbial Cells 574
Disrupting Microbial Cells 576
Downstream Processing 578
Inclusion Bodies 579
Utilizing an Immobilized Enzyme 582
Magnetic Separation of Proteins 582
Large-Scale Production of DNA and RNA 583
Plasmid DNA 583
mRNA 586
Summary 587
References 587
Review Questions 590
10. Genetic Engineering of Plants: Methodology 591
Plant Transformation with the Ti Plasmid of A. tumefaciens 595
Ti Plasmid-Derived Vector Systems 597
Increasing Transformation Efficiency 601
Microprojectile Bombardment 603
Chloroplast Engineering 604
Very-High-Level Protein Expression 607
Use of Reporter Genes in Transformed Plant Cells 610
Manipulation of Gene Expression in Plants 611
Transient Gene Expression 611
Plant Promoters 616
Manipulation of Genes in Plants 617
Facilitating Protein Purification 621
Protein Glycosylation 623
Gene Stacking 624
CRISPR-Based Directed Evolution 625
Polycistronic Gene Expression 626
Production of Marker-Free Transgenic Plants 626
Removing Marker Genes from Nuclear DNA 627
Removing Marker Genes from Chloroplast DNA 632
Summary 633
References 634
Review Questions 636
11. Transgenic Plants 637
Insect Resistance 637
Bacillus thuringiensis Insecticidal Toxin 637
Increasing Expression of the B. thuringiensis Protoxin 642
Other Strategies for Protecting Plants against Insects 645
Preventing the Development of B. thuringiensis-Resistant Insects 652
Targeting Aphids 657
Virus Resistance 658
Viral Coat Protein-Mediated Protection 658
Protection by Expression of Other Genes 663
Herbicide Resistance 668
Glyphosate 669
Dicamba 672
Other Herbicides 673
Fungus and Bacterium Resistance 674
Transgenic Plants 675
RNAi and CRISPR/Cas 681
Salt and Drought Stress 682
Increasing Trehalose Production 683
Sequestering Sodium Ions 684
Delaying Drought-Induced Senescence 685
Phytoremediation 686
Fruits and Flowers 688
Flavr Savr Tomato 688
Lowering Ethylene Levels 688
CRISPR Mutants 690
Modification of Plant Nutritional Content 690
Amino Acids 690
Lipids 692
Vitamins 695
Iron 698
Gluten 700
Modification of Food Plant Taste and Appearance 701
Preventing Discoloration 701
Starch 703
Plants as Bioreactors 706
Antibodies 706
Pharmaceuticals and Vaccines 709
Poly(3-Hydroxybutyric Acid) 710
Edible Vaccines 711
Edible Cholera Vaccines 712
Edible E. coli Vaccines 714
Plant Yield 716
Increasing Grain Yield 716
Increasing Harvest Index 716
Decreasing Lignin Content 717
Decreasing Pectin Content 720
Increasing Oxygen Content 722
Summary 723
References 724
Review Questions 729
12. Transgenic Animals 731
Transgenic Animal Methodologies 733
DNA Microinjection Method 733
Retroviral Vector Method 736
Engineered Embryonic Stem Cell Method 737
Somatic Cell Nuclear Transfer for Transgenic Livestock 743
Genome Editing with the CRISPRCas System 744
Conditional Gene Modification with the CreloxP Recombination System 747
Control of Transgene Expression with the TetracyclineInducible System 749
Gene Knockdown by RNA Interference 754
Transgenic Animal Models of Human Diseases 756
Mouse Models of Alzheimers Disease 756
Mouse Model of Duchenne Muscular Dystrophy 759
Rabbit Models of Cardiovascular Disease 761
Zebrafish Melanoma Model 763
Nonhuman Primate Models of Neurodevelopmental
Disorders 766
Animal Bioreactors for Production of Recombinant Therapeutic Proteins 767
Production of Recombinant Antithrombin in Goat Milk 768
Production of a Human Protease Inhibitor in Rabbits 770
Production of Therapeutic Proteins in Chicken Eggs 771
Production of Donor Organs in Pigs 773
Enhancing Production Traits of Food Animals 774
DiseaseResistant Livestock 774
Improving Milk Quality 781
Increasing Muscle Mass in Cattle 782
Enhancing Growth of Salmon 786
Gene Drives To Eradicate VectorTransmitted Diseases 787
Malaria Vector Population Suppression 789
Dengue Fever VirusResistant Mosquitoes 791
Reversal Drives 792
Summary 795
References 796
Review Questions 797
13. Molecular Biotechnology and Society 799
Development of Guidelines for Recombinant DNA Research 800
Deliberate Release of Genetically Modified Microorganisms 802
Environmental Concerns 802
Regulations 803
Regulation of Genetically Modified Foods 804
Food Ingredients Produced by Genetically Engineered Microorganisms 804
Genetically Modified Crops 807
Genetically Engineered Livestock 810
Societal Concerns about Genetically Modified Foods 812
Alteration of Nutritional Content of Food 812
Potential for Introducing Toxins or Allergens into Food 816
Potential for Transferring Transgenes from Food to Humans or Intestinal Microorganisms 819
Controversy about the Labeling of Genetically Modified Foods 820
Impact of Genetically Engineered Crops on Biodiversity 822
Who Benefits from the Production of Genetically Modified Foods? 824
Environmental Benefits of Genetically Modified Crops 825
How Do Views about Genetically Engineered Organisms Impact Trade? 827
Regulation and Safety of Medical Products of Biotechnology 827
New Biological Drugs 828
Genetic and Genomic Testing 832
Economic Issues 835
Patenting Biotechnology 837
Patenting 838
Patenting in Different Countries 839
Patenting Nucleic Acid Sequences 841
Patenting Living Organisms 842
Patenting and Fundamental Research 844
Summary 845
References 846
Review Questions 848
Amino Acids of Proteins and Their Designations 851
Index 853
