Molecular Biotechnology

The Development of Molecular Biotechnology 1


Emergence of Molecular Biotechnology 1


Recombinant DNA Technology 3


Commercialization of Molecular Biotechnology 4


Concerns and Consequences 7


SUMMARY


9


REFERENCES 9


REVIEW


QUESTIONS


10


Fundamental Technologies 11


Molecular Cloning 11


Preparation of DNA for Cloning 11


Insertion of Target DNA into a Plasmid Vector 16


Transformation and Selection of Cloned DNA in a Bacterial Host 20


Cloning Eukaryotic Genes 24


Recombinational Cloning 28


Genomic Libraries 30


Genome Engineering using


CRISPR Technology 32


Polymerase Chain Reaction 35


Amplification of DNA by PCR 36


Cloning PCR Products 39


Quantitative PCR 39


Chemical Synthesis of DNA 42


Synthesis of Oligonucleotides 42


Assembling Oligonucleotides into Genes 48


Gene Synthesis by PCR 50


DNA Sequencing Technologies 50


Dideoxynucleotide Procedure 53


Pyrosequencing 55


Sequencing using Reversible Chain Terminators 57


Sequencing by Single Molecule Synthesis 57





Sequencing Whole Genomes 59


Preparation of Genomic DNA Sequencing Libraries 60


High-Throughput Next-Generation Sequencing Strategies 61


Genome Sequence Assembly 63


Sequencing Metagenomes 64


Genomics 64


Transcriptomics 67


Proteomics 72


Metabolomics 85


SUMMARY


87


REFERENCES 89


REVIEW


QUESTIONS


91


Production of Recombinant Proteins 93


Protein Production in Prokaryotic Hosts 93


Regulation of Transcription 94


Increasing Translation Efficiency 98


Increasing Protein Stability 102


Increasing Protein Secretion 106


Facilitating Protein Purification 110


DNA Integration into the Host Chromosome 115


Heterologous Protein Production in Eukaryotic Cells 120


Posttranslational Modification of Eukaryotic Proteins 120


General


Features of Eukaryotic Expression Systems 122


Yeast Expression Systems 124


Baculovirus-Insect Cell Expression Systems 136


Mammalian Cell Expression Systems 143


Protein Engineering 153


Directed Mutagenesis 154


Random Mutagenesis 158


Examples of Protein Engineering 162


SUMMARY


171


REFERENCES 173


REVIEW


QUESTIONS


175


Molecular Diagnostics 177


Immunological Approaches to Detect Protein Biomarkers 178


Antibodies 178


Agglutination 183


Enzyme-Linked Immunosorbent Assays 183


Protein Arrays to Detect Polygenic Diseases 189


Immunoassays for Protein Conformation-Specific Disorders 191


DNA-Based Diagnostic Approaches 193


Hybridization Probes 193


PCR-Based Detection Methods 200


DNA Microarrays 208


Whole Genome Sequencing to Assess Genetic Disease Risk 214


Detecting RNA Signatures of Disease 215


Detection of Disease-Associated Changes in Gene Expression


Using Microarrays 215


Detection of RNA Signatures of Antibiotic Resistance in Bacteria 216


Detection of miRNA Signatures of Cancers 217


Biofluorescent and Bioluminescent Systems 219


Fluorescent Proteins


219


Luciferase 221


Microbial Biosensors 222


SUMMARY


224


REFERENCES 225


REVIEW


QUESTIONS


227


Protein Therapeutics 229


Pharmaceuticals 230


Human Interferons 231


Human Growth Hormone 235


Tumor Necrosis Factor Alpha 237


Extending Protein Half-Life 238


Enzymes 240


DNase


I 240


Alginate Lyase 242


Phenylalanine Ammonia Lyase 245


1-Antitrypsin 247


Glycosidases 248


Masking Nonhuman Epitopes 249


Engineering Bacteriophages 250


Targeting Mitochondria 253


Lactic Acid Bacteria 255


Interleukin-10 255


Leptin 258


An HIV Inhibitor 258


Insulin 260


Recombinant Antibodies 261


Hybrid Human-Mouse Monoclonal Antibodies 264


Human Monoclonal Antibodies 268


Antibody Fragments 270


Combinatorial Libraries of Antibody Fragments 274


A Combinatorial Library of Full-Length Antibodies 277


Shuffling CDR Sequences 278


Dual-Variable-Domain Antibodies 280


Anticancer Antibodies 281


Antibodies Against Various Diseases 284


Antiobesity Antibodies 287


Enhanced Antibody Half-Life 290


SUMMARY


292


REFERENCES 292


REVIEW


QUESTIONS


295


Nucleic Acids as Therapeutic Agents 297


Targeting Specific mRNA and DNA Sequences


299


Antisense RNA 299


Aptamers 302


Ribozymes and DNAzymes 307


Interfering RNA 311


Zinc Finger Nucleases 315


CRISPR-Cas System 317


Nanozymes 318


Nanoparticles 319


Viral Delivery Systems 319


Nonviral Delivery Systems 325


Direct Injection 325


Lipids 327


Bacteria 328


Dendrimers 331


Antibodies 332


Aptamers 332


Transposons 334


Gene Therapy 335


Prodrug Activation Therapy 335


Promoterless Gene Targeting 337


SUMMARY


338


REFERENCES 339


REVIEW


QUESTIONS


341


Vaccines 343


Vaccination 343


Current and Future Vaccines 345


Subunit Vaccines 347


Herpes Simplex Virus 348


Cholera 350


SARS 350


Staphylococcus aureus


351


Human Papillomavirus 353


Foot-and-Mouth Virus 354


Streptococcus 356


Delivery 357


Peptide Vaccines 359


Malaria 359


Genetic Immunization: DNA Vaccines 363


Delivery 364


Cancer 370


Dental Caries 370


Attenuated Vaccines 372


Herpes Simplex Virus 372


Cholera 374


Salmonella Species 375


Leishmania Species 378


Vector Vaccines 378


Vaccines Directed against Viruses 378


Vaccines Directed against Bacteria 388


Bacteria as Antigen Delivery Systems 392


Monoclonal Antibody Passive Immunity 396


Influenza Virus 396


SUMMARY


397


REFERENCES 398


REVIEW


QUESTIONS


400


Industrial and Environmental Uses of


Recombinant Microorganisms 403


Restriction Endonucleases 403


Small Biological Molecules 405


L-Ascorbic Acid 407


Indigo 410


Amino Acids 412


Lycopene 417


Antibiotics 418


Biopolymers 429


Microbial Degradation of Xenobiotics 434


Genetic Engineering of Biodegradative Pathways 436


Utilization of Starch and Sugars 445


Commercial Production of Fructose and Alcohol 446


Increasing Alcohol Production 448


Improving Fructose Production 453


Utilization of Cellulose and Hemicellulose 454


Lignocellulosics 455


Cellulase Genes 457


Direct Conversion of Biomass to Ethanol 462


Zymomonas mobilis 464


Lipids from Cyanobacteria 467


Hydrogen Production 468


SUMMARY


470


REFERENCES 471


REVIEW


QUESTIONS


474


Large-Scale Production of Proteins from


Recombinant Microorganisms 475


Principles of Microbial Growth 476


Batch Fermentation 477


Fed-Batch Fermentation 479


Continuous Fermentation 480


Maximizing The Efficiency of The Fermentation Process 481


High-Density Cell Cultures 483


Increasing Plasmid Stability 484


Quiescent E. Coli Cells 485


Protein Secretion 486


Reducing Acetate 489


Bioreactors 491


Typical Large-Scale Fermentation Systems 494


Two-Stage Fermentation in Tandem Airlift Reactors 495


Two-Stage Fermentation in a Single Stirred-Tank Reactor 496


Batch versus


Fed-Batch Fermentation 498


Harvesting Microbial Cells 501


Disrupting Microbial Cells 502


Downstream Processing 504


Protein Solubilization 506


Utilizing an Immobilized Enzyme 507


Magnetic Separation of Proteins 507


Large-Scale Production of Plasmid DNA 508


SUMMARY 511


REFERENCES 512


REVIEW QUESTIONS 514


10 Genetic Engineering of Plants: Methodology 515


Plant Transformation with the Ti Plasmid of


A. Tumefaciens 516


Ti Plasmid-Derived Vector Systems 522


Microprojectile Bombardment 526


Chloroplast Engineering 527


Very High Level Protein Expression 529


Use of Reporter Genes in Transformed Plant Cells 532


Manipulation of Gene Expression in Plants 533


Transient Gene Expression 533


Plant Promoters 536


Targeted Gene Editing 538


Facilitating Protein Purification 539


Protein Glycosylation 541


Production of Marker-Free Transgenic Plants 542


Removing Marker Genes from Nuclear DNA 543


Removing Marker Genes from Chloroplast DNA 545


SUMMARY 546


REFERENCES 547


REVIEW QUESTIONS 549


11 Transgenic Plants 551


Insect Resistance 551


B. thuringiensis Insecticidal Toxin 551


Increasing Expression of the B. thuringiensis Protoxin 555


Other Strategies for Protecting Plants against Insects 558


Preventing the Development of B. thuringiensis-Resistant


Insects 564


Targeting Aphids 569


Virus Resistance 570


Viral Coat Protein-Mediated Protection 570


Protection by Expression of Other Genes 574


Herbicide Resistance 578


Fungus and Bacterium Resistance 583


Salt and Drought Stress 588


Fruit Ripening and Flower Wilting 592


Modification of Plant Nutritional Content 594


Amino Acids 594


Lipids 595


Vitamins 599


Iron 601


Gluten 602


Modification of Food Plant Taste and Appearance 603


Preventing Discoloration 603


Starch 605


Plants as Bioreactors 608


Antibodies 608


Poly(3-hydroxybutyric Acid) 610


Eddible Vaccines 611


Plant Yield 615


Altering Lignin Content 615


Increasing Oxygen Content 618


SUMMARY


619


REFERENCES 620


REVIEW


QUESTIONS


624


12 Transgenic Animals 625


Transgenic Mice: Methodology 626


DNA Microinjection Method 627


Retroviral Vector Method 629


Engineered Embryonic Stem Cell Method 631


Conditional Gene Inactivation with the Cre-loxP


Recombination System 637


Genome Editing with the CRISPR-Cas System


641


Gene Knockdown by RNA Interference 643


Transgenic Mice: Applications 644


Transgenic Disease Models: Alzheimer Disease 644


Transgenic Mice as Test Systems 647


Control of Transgene Expression 651


Conditional Control of Cell Death 654


Transgenic Livestock 656


Cloning Livestock by Somatic Cell Nuclear Transfer 656


Production of Pharmaceuticals 658


Production of Donor Organs 660


Disease Resistant Livestock 661


Improving Milk Quality 664


Improving Animal Production Traits 665


Transgenic Poultry 669


Transgenic Fish 673


SUMMARY 676


REFERENCES 676


REVIEW QUESTIONS 678


13 Molecular Biotechnology and Society 679


Development of Guidelines for Recombinant


DNA Research 680


Deliberate Release of Genetically Modified


Microorganisms 682


Environmental Concerns 682


Regulations 683


Regulation of Genetically Modified Foods 684


Food Ingredients Produced by Genetically Engineered


Microorganisms 684


Genetically Modified Crops 687


Genetically Engineered Livestock 691


Societal Concerns About Genetically Modified Foods 692


Alteration of Nutritional Content of Food 692


Potential for Introducing Toxins or Allergens into Food 696


Potential for Transferring Transgenes from Food to Humans


or Intestinal Microorganisms 698


Controversy About the Labeling of Genetically Modified Foods 700


Impact of Genetically Engineered Crops on Biodiversity 700


Who Benefits from Production of Genetically Modified Foods? 703


Environmental Benefits of Genetically Modified Crops 704


How do Views about Genetically Engineered Organisms


Impact Trade? 705


Regulation and Safety of Medical Products of


Biotechnology 706


New Biological Drugs 706


Genetic and Genomic Testing 709


Economic Issues 711


Patenting Biotechnology 714


Patenting 714


Patenting in Different Countries 716


Patenting Nucleic Acid Sequences 717


Patenting Living Organisms 719


Patenting and Fundamental Research 720


SUMMARY


721


REFERENCES 722


REVIEW


QUESTIONS


724


Amino Acids of Proteins and Their Designations 725


Index 727