Molecular Biotechnology Molecular Biotechnology Principles and Applications of Recombinant DNA SIXTH EDITION An authoritative introduction to the fast-changing world of molecular biotechnology In continuous publication since 1994 and now in its sixth edition, Molecular Biotechnology: Principles and Applications of Recombinant DNA has been effective in introducing this complex field to students for more than 25 years. This textbook covers essentially every aspect of the field of molecular biotechnology, which is constantly changing and adapting in light of new advances. This edition includes the latest techniques in DNA sequencing and genetic engineering of microbial, plant, and animal genomes, including human genome editing, as well as updates across many areas, such as: * Immunological assays for disease diagnosis, more effective bacteriophage therapy, and new ways of dealing with antibiotic-resistant bacteria * New and developing vaccines for influenza, tuberculosis, and emerging viral threats, including Zika and SARS-CoV-2 * Engineering bacteria to perform plastic degradation and green algae to produce hydrogen, altering amino acid biosynthesis, and creating designer cellulosomes * Production of humanized monoclonal antibodies in plants, modifying hybrid plants to produce clonal hybrids, and protecting plants from viral and fungal diseases Molecular Biotechnology features nearly 600 detailed figures and is an ideal textbook for undergraduate and graduate courses in introductory biotechnology, as well as courses dedicated to utilizing this technology, such as medical, agricultural, environmental, and industrial biotechnology applications.

About the Authors Bernard R. Glick, PhD, is a distinguished professor emeritus at the University of Waterloo, Ontario, Canada, where he was the chair of biology from 2002 to 2008. He has authored more than 400 publications that have garnered over 54,000 citations. Cheryl L. Patten, PhD, is a professor of microbiology in the Biology Department at the University of New Brunswick, Fredericton, Canada. She teaches courses in introductory biology, microbiology, and biotechnology.

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 alpha1 -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