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

Single‐Molecule Real‐Time Sequencing 55

Nanopore Sequencing 56

Sequencing Whole Genomes 56

Preparation of Genomic DNA Sequencing Libraries 57

High‐Throughput Next‐Generation 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

Baculovirus–Insect 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

Enzyme‐Linked Immunosorbent Assays 186

Protein Arrays To Detect Polygenic Diseases 194

Immunoassays for Protein Conformation‐Specific Disorders 197

DNA‐Based Diagnostic Approaches 199

Hybridization Probes 199

PCR‐Based Detection Methods 207

CRISPR‐Cas‐Based Diagnostic Assays 218

DNA Microarrays 219

Whole‐Genome Sequencing To Assess Genetic Disease Risk 225

Detecting RNA Signatures of Disease 226

Detection of Disease‐Associated 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

α1 -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

Parkinson’s Disease 279

Cancer and Bacteria 279

Recombinant Antibodies 280

Hybrid Human–Mouse 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

CRISPR‐Cas 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 CRISPR‐Cas System 744

Conditional Gene Modification with the Cre–loxP Recombination System 747

Control of Transgene Expression with the Tetracycline‐Inducible System 749

Gene Knockdown by RNA Interference 754

Transgenic Animal Models of Human Diseases 756

Mouse Models of Alzheimer’s 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

Disease‐Resistant Livestock 774

Improving Milk Quality 781

Increasing Muscle Mass in Cattle 782

Enhancing Growth of Salmon 786

Gene Drives To Eradicate Vector‐Transmitted Diseases 787

Malaria Vector Population Suppression 789

Dengue Fever Virus‐Resistant 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