Introduction to Molecular Biotechnology

Michael Wink studied biology and chemistry in Bonn and was awarded his doctorate from TU Braunschweig in 1980. After gaining his lecturing qualification in 1984/1985, he was awarded a Heisenberg grant by the German Research Council to work at the Max Planck Institute for Breeding Research in Cologne and from then at the Gene Center of Ludwig-Maximilians University in Munich. Following a chair for Pharmaceutical Biology at Mainz University in 1988, he accepted the post of Professor for Pharmaceutical Biology at the University of Heidelberg one year later. His areas of interest include pharmaceutical research, molecular biotechnology, and medicinal plants, as well as research into natural products and evolution.

• Abbreviations xixPart I Fundamentals of Cellular and Molecular Biology 11 The Cell as the Basic Unit of Life 3Michael WinkReferences 8Further Reading 82 Structure and Function of Cellular Macromolecules 9Michael Wink2.1 Structure and Function of Sugars 92.2 Structure of Membrane Lipids 132.3 Structure and Function of Proteins 172.4 Structure of Nucleotides and Nucleic Acids (DNA and RNA) 25References 32Further Reading 323 Structure and Functions of a Cell 33Michael Wink3.1 Structure of a Eukaryotic Cell 333.1.1 Structure and Function of the Cytoplasmic Membrane 333.1.1.1 Membrane Permeability 333.1.1.2 Transport Processes Across Biomembranes 343.1.1.3 Receptors and Signal Transduction at Biomembranes 373.1.2 Endomembrane System in a Eukaryotic Cell 403.1.3 Mitochondria and Chloroplasts 453.1.4 Cytoplasm 493.1.5 Cytoskeleton 513.1.6 Cell Walls 533.2 Structure of Bacteria 533.3 Structure of Viruses 553.4 Differentiation of Cells 563.5 Cell Death 60References 61Further Reading 614 Biosynthesis and Function of Macromolecules (DNA, RNA, and Proteins) 63Michael Wink4.1 Genomes, Chromosomes, and Replication 634.1.1 Genome Size 634.1.2 Composition and Function of Chromosomes 674.1.3 Mitosis and Meiosis 694.1.4 Replication 714.1.5 Mutations and Repair Mechanisms 724.2 Transcription: From Gene to Protein 774.3 Protein Biosynthesis (Translation) 81Further Reading 855 Distributing Proteins in the Cell (Protein Sorting) 87Michael Wink5.1 Import and Export of Proteins via the Nuclear Pore 875.2 Import of Proteins in Mitochondria, Chloroplasts, and Peroxisomes 885.3 Protein Transport into the Endoplasmic Reticulum 895.4 Vesicle Transport from the ER via the Golgi Apparatus to the Cytoplasmic Membrane 92References 94Further Reading 946 Evolution and Diversity of Organisms 95Michael Wink6.1 Prokaryotes 956.2 Eukaryotes 95References 101Further Reading 101Part II Standard Methods in Molecular Biotechnology 1037 Isolation and Purification of Proteins 105Thomas Wieland7.1 Introduction 1057.2 Producing a Protein Extract 1067.3 Gel Electrophoretic Separation Methods 1077.3.1 Principles of Electrophoresis 1077.3.2 Native Gel Electrophoresis 1077.3.3 Discontinuous Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 1077.3.4 Two-Dimensional (2D) Gel Electrophoresis and Isoelectric Focusing (IEF) 1087.3.5 Detecting Proteins in Gels 1087.4 Methods of Protein Precipitation 1097.5 Column Chromatography Methods 1097.5.1 General Principles of Separation 1097.5.1.1 Size Exclusion Chromatography (Gel Filtration) 1097.5.1.2 Hydrophobic Interaction Chromatography 1117.5.1.3 Ion Exchange Chromatography 1117.5.1.4 Hydroxyapatite Chromatography 1127.5.2 Group-Specific Separation Techniques 1127.5.2.1 Chromatography on Protein A or Protein G 1127.5.2.2 Chromatography on Cibacron Blue (Blue Gel) 1127.5.2.3 Chromatography on Lectins 1127.5.2.4 Chromatography on Heparin 1137.5.3 Purification of Recombinant Fusion Proteins 1137.5.3.1 Chromatography on Chelating Agents 1137.5.3.2 Chromatography on Glutathione Matrices 1147.6 Examples 1147.6.1 Example 1: Purification of Nucleoside Diphosphate Kinase from the Cytosol of Bovine Retina Rod Cells 1147.6.2 Example 2: Purification of Recombinant His6-RGS16 After Expression in E. coli 114Further Reading 1158 Mass Spectrometry and Applications in Proteomics and Microbial Identification 117Andreas Schlosser and Wolf D. Lehmann8.1 Principles of ESI and MALDI Mass Spectrometry 1178.2 Instrumental Setup 1188.3 Intact Protein Analysis 1198.3.1 Protein Digestion 1198.3.2 Peptide Fragmentation 1198.3.3 Protein Identification with MS/MS Spectra 1218.4 Protein and Proteome Quantification 1218.4.1 Label-Free Quantification 1218.4.2 Chemical Stable Isotope Labeling 1218.4.3 Metabolic Stable Isotope Labeling 1228.5 Protein–Protein Interaction Analysis 1238.6 Analysis of Posttranslational Modifications 1248.7 Microbial Identification and Resistance Detection 125References 1269 Isolation of DNA and RNA 129Hans Weiher9.1 Introduction 1299.2 DNA Isolation 1299.3 RNA Isolation 1319.3.1 Enrichment of mRNA 131Reference 13110 Chromatography and Electrophoresis of Nucleic Acids 133Hans Weiher10.1 Introduction 13310.2 Chromatographic Separation of Nucleic Acids 13310.3 Electrophoresis 13410.3.1 Agarose Gel Electrophoresis: Submarine Electrophoresis 13410.3.2 Pulsed-Field Agarose Gel Electrophoresis 13410.3.3 Polyacrylamide Gel Electrophoresis (PAGE) 135Further Reading 13511 Hybridization of Nucleic Acids 137Hans Weiher11.1 Significance of Base Pairing 13711.2 Experimental Hybridization: Kinetic and Thermodynamic Control 13711.3 Analytical Techniques 13811.3.1 Clone Detection, Southern Blotting, Northern Blotting, and Gene Diagnosis 13811.3.2 Systematic Gene Diagnosis and Expression Screening Based on Gene Arrays 13911.3.3 In Situ Hybridization 139References 140Further Reading 14012 Use of Enzymes in the Modification of Nucleic Acids 141Ingrid Herr and MichaelWink12.1 Restriction Enzymes (Restriction Endonucleases) 14112.2 Ligases 14212.3 Methyl transferases 14212.4 DNA Polymerases 14312.5 RNA Polymerases and Reverse Transcriptase 14412.6 Nucleases 14412.7 T4 Polynucleotide Kinase 14412.8 Phosphatases 145Further Reading 14513 Polymerase Chain Reaction 147Richard Jäger and Hans Weiher13.1 Introduction 14713.2 PCR Methods 14713.2.1 Basic Principle 14713.2.2 Primer Design and Hot Start PCR 14813.2.3 Multiplex PCR 14913.2.4 RT-PCR 14913.2.5 Qualitative Analysis of the PCR Products 14913.3 PCR as a Quantitative Method 14913.3.1 PCR Phases and PCR Efficiency 14913.3.2 Quantitative Real-Time PCR 15013.3.3 Digital PCR 15113.4 Areas of Application 15113.4.1 Genome Analysis 15113.4.2 Cloning Techniques 15213.4.3 Gene Expression Studies 152Further Reading 15214 DNA Sequencing 153Richard Jäger and HansWeiher14.1 Introduction 15314.2 The Sanger Method 15314.3 Pyrosequencing 15414.4 Second-Generation Sequencing: Illumina and Ion Torrent 15514.4.1 Overview 15514.4.2 The Illumina Sequencing System 15514.4.3 The Ion Torrent Sequencing System 15614.5 Third-Generation Sequencing Techniques 15614.5.1 Overview 15614.5.2 SMRT Sequencing 15714.5.3 Nanopore Sequencing 15714.6 The Impact of the DNA Sequencing Technology 158References 158Further Reading 158Websites 15815 Cloning Procedures 159Thomas Wieland and Susanne Lutz15.1 Introduction 15915.2 Construction of Recombinant Vectors 15915.2.1 Insert 15915.2.2 Vector 16115.2.3 Essential Components of Vectors 16215.2.3.1 Bacterial Origin of Replication (ori) 16215.2.3.2 Antibiotic Resistance 16215.2.3.3 Polylinkers 16215.2.4 Cloning Using Recombination Systems 16215.2.5 Further Components of Vectors for Prokaryotic Expression Systems 16315.2.5.1 Promoter 16315.2.5.2 Ribosome-Binding Site 16315.2.5.3 Termination Sequence 16415.2.5.4 Fusion Sequence 16415.2.6 Further Components of Eukaryotic Expression Vectors 16415.2.6.1 Eukaryotic Expression Vectors: Yeast 16415.2.6.2 Eukaryotic Expression Vectors for Mammal Cells 16515.2.6.3 Viral Expression Systems for Mammalian Cells 16715.2.7 Nonviral Introduction of Heterologous DNA to Host Organisms (Transformation, Transfection) 16815.2.7.1 Transformation of Prokaryotes 16815.2.7.2 Transformation of Yeast Cells 16915.2.7.3 Transfection of Mammal Cells 169Further Reading 17016 Expression of Recombinant Proteins 171Thomas Wieland16.1 Introduction 17116.2 Expression of Recombinant Proteins in Host Organisms 17116.2.1 Expression in E. coli 17216.2.2 Expression in Yeasts 17516.2.3 Expression in Insect Cells 17716.2.3.1 Expression Based on Recombinant Baculoviruses 17716.2.3.2 Expression of Proteins in Stably Transfected Insect Cells 17816.2.4 Expression of Proteins in Mammalian Cells 17816.3 Expression in Cell-Free Systems 17916.3.1 Expression of Proteins in Reticulocyte Lysates 18016.3.2 Protein Expression Using E. coli Extracts 180Further Reading 18017 Patch Clamp Method 181Robert Kraft17.1 Ion Channels 18117.2 Technical Requirements of the Patch Clamp Method 18117.3 Patch Clamp Configurations 18217.4 Applications of the Patch Clamp Method 183Reference 185Further Reading 18518 Cell Cycle Analysis 187Stefan Wölfl18.1 Introduction 18718.2 Analyzing the Cell Cycle 18718.3 Experimental Analysis of the Cell Cycle 18918.3.1 Preparing Synchronized Cell Cultures of S. cerevisiae 18918.3.1.1 Centrifugal Elutriation 19018.3.1.2 Cell Cycle Arrest Using α-Factor 19018.3.2 Identification of Cell Cycle Stages 19118.3.2.1 Budding Index 19118.3.2.2 Fluorescent Staining of the Nucleus 19118.3.2.3 Detection of Cell Cycle Phases Using Fluorescent Proteins as Reporters 194Acknowledgments 195Further Reading 19619 Microscopic Techniques 197Stephan Diekmann19.1 Introduction 19719.2 Electron Microscopy 19719.2.1 Cryo-electron Microscopy 19919.2.2 Electron Tomography 19919.3 Atomic or Scanning Force Microscopy 19919.3.1 Force Spectroscopy 20019.3.2 Advantages and Disadvantages 20119.4 Light Microscopy 20119.4.1 Deconvolution 20219.4.2 Confocal Microscopy 20219.4.3 Why Fluorescence? 20319.4.4 Nanoscopy 20319.5 Microscopy in the Living Cell 20419.5.1 Analysis of Fluorescently Labeled Proteins In Vivo 20519.5.2 Fluorescence Recovery After Photobleaching 20619.5.3 Fluorescence Correlation Spectroscopy 20619.5.4 Förster Resonance Energy Transfer and Fluorescence Lifetime Imaging Microscopy 20719.5.5 Single-Molecule Fluorescence 207Further Reading 20720 Laser Applications 209Rainer Fink20.1 Laser Development: A Historical Perspective 20920.2 Types of Lasers and Setups 21020.3 Properties of Laser Radiation 21020.4 Applications 21120.4.1 Laser Scanning Microscopy 21120.4.2 Optical Tweezers 21220.4.3 Laser Microdissection and Laser Therapy 21220.4.4 Manufacturing of Products in Medical Technology and Biotechnology Products 213Further Reading 213Part III Key Topics 21521 Sequencing the Universe of Life 217Stefan Wiemann21.1 What to Sequence? 21721.1.1 Whole-Genome Sequencing 21721.1.2 Exome Sequencing 22021.1.3 (Gene) Panel Sequencing 22021.1.4 RNA Sequencing 22121.1.4.1 Tag- vs. Full-Length Sequencing 22121.1.4.2 Sequencing of RNA Species and Modifications 22121.1.4.3 Sequencing of Single Cells 22221.1.4.4 In Situ Sequencing 22221.1.5 (Whole-Genome) Bisulfite Sequencing of DNA 22321.1.6 Sequencing to Characterize Chromatin Structure and Beyond 22321.2 Sequencing Projects: Human 22421.2.1 Initial Sequencing of the Human Genome 22421.2.2 The 1000 Genomes Project: Assessing Natural Variation 22421.2.3 Screening for Genetic Disease 22521.2.4 Sequencing of Populations 22621.2.5 TCGA and ICGC: Screening for Cancer Driver Mutations 22621.3 Sequencing Other Species, Environments,… 22821.4 Sequencing in the Clinics: Personalizing Oncology 22821.5 Sequencing in the Private Sector: Direct to Consumer Testing (DTC) 23121.6 The Information Content of a Genome Sequence and Ethical Consequences 231References 23222 Cellular Systems Biology 239Melanie Boerries, Hauke Busch, and Rainer König22.1 Introduction 23922.2 Analysis of Cellular Networks by Top-Down Approaches 24022.2.1 Motivation 24022.2.2 Definitions and Construction of the Networks 24022.2.3 Gene Set Enrichment Tests 24122.2.4 Inferring Gene Regulators Employing Gene Regulatory Models 24222.2.5 Network Descriptors 24322.2.5.1 Scale-Free Networks 24322.2.5.2 Centrality 24322.2.5.3 The Clustering Coefficient 24422.2.6 Detecting Essential Enzymes with a Machine Learning Approach 24422.2.7 Elementary Flux Modes 24422.3 Overview over Bottom-Up Modeling of Biochemical Networks 24722.3.1 Motivation 24722.3.2 Choosing Model Complexity and Model Building 24822.3.3 Model Simulation 25122.3.4 Model Calibration 25222.3.5 Model Verification and Analysis 25422.3.6 Examples 254Further Reading 258References 25923 Protein–Protein and Protein–DNA Interactions 261Peter Uetz and Ehmke Pohl23.1 Protein–Protein Interactions 26123.1.1 Classification and Specificity: Protein Domains 26123.1.2 Protein Networks and Complexes 26223.1.3 Structural Properties of Interacting Proteins 26223.1.4 Which Forces Mediate Protein–Protein Interactions? 26323.1.4.1 Thermodynamics 26423.1.4.2 Energetics 26423.1.5 Methods to Examine Protein–Protein Interactions 26423.1.6 Theoretical Prediction of Protein–Protein Interactions 26623.1.7 Regulation of Protein–Protein Interactions 26623.1.8 Biotechnological and Medical Applications of Protein–Protein Interactions 26823.2 Protein–DNA Interactions 26923.2.1 Specific Protein–DNA Interaction 26923.2.2 Thermodynamic Consideration 27023.2.3 Methods to Study Protein–DNA Interactions 27023.2.3.1 Structural Classification of Protein–DNA Complexes 27023.2.4 Regulatory Networks and System Biology 27023.2.5 Medical Importance of Protein–DNA Interactions 27323.2.6 Biotechnological Applications 274References 275Further Reading 27524 Bioinformatics 277Benedikt Brors24.1 Introduction 27724.2 Data Sources 27724.2.1 Primary Databases: EMBL/GenBank/DDBJ, PIR, and Swiss-Prot 27724.2.2 Genome Databases: Ensembl and GoldenPath 27824.2.3 Motif Databases: BLOCKS, PROSITE, Pfam, ProDom, and SMART 27824.2.4 Molecular Structure Databases: PDB and SCOP 27824.2.5 Transcriptome Databases: SAGE, ArrayExpress, and GEO 27924.2.6 Reference Databases: PubMed, OMIM, and GeneCards 27924.2.7 Pathway Databases and Gene Ontology 27924.3 Sequence Analysis 28024.3.1 Kyte–Doolittle Plot, HelicalWheel Analysis, and Signal Sequence Analysis 28024.3.2 Pairwise Alignment 28124.3.2.1 Local/Global 28124.3.2.2 Optimal/Heuristic 28224.3.3 Alignment Statistics 28224.3.4 Multiple Alignment 28224.4 Evolutionary Bioinformatics 28324.4.1 StatisticalModels of Evolution 28324.4.2 Relation to Score Matrices 28424.4.3 Phylogenetic Analysis 28524.5 Gene Prediction 28524.5.1 Neural Networks or HMMs Based on Hexanucleotide Composition 28624.5.2 Comparison with Expressed Sequence Tags or Other Genomes (Fugu, Mouse) 28624.6 Bioinformatics in Transcriptome and Proteome Analysis 28724.6.1 Preprocessing and Normalization 28724.6.2 Feature Selection 28824.6.3 Similarity Measures: Euclidean Distance, Correlation, Manhattan Distance, Mahalanobis Distance, and Entropy Measures 28824.6.4 Unsupervised Learning Procedures: Clustering, Principal Component Analysis, Multidimensional Scaling, and Correspondence Analysis 28924.6.5 Supervised Learning Procedures: Linear Discriminant Analysis, Decision Trees, Support Vector Machines, and ANNs 28924.6.6 Analysis of Overrepresentation of Functional Categories 29024.7 Analysis of Ultraparallel Sequencing Data 29124.7.1 Mapping of Ultraparallel Sequencing Data 29124.7.2 Genome (Re-)sequencing 29224.7.3 Transcriptome Sequencing 29224.7.4 ChIP-seq 29324.7.5 Epigenetic Analysis 29324.7.6 Single-Cell Analysis 29424.7.7 Bioethics of Human Sequencing Data 29424.8 Bioinformatic Software 294Further Reading 29525 Drug Research 297Manfred Koegl, Ralf Tolle, Ulrich Deuschle, Claus Kremoser, and Michael Wink25.1 Introduction 29725.2 Active Compounds and Their Targets 29725.2.1 Identification of Potential Targets in the Human Genome 29825.2.2 Comparative Genome Analysis 29825.2.3 Experimental Target Identification: In Vitro Methods 29925.2.4 Experimental Identification of Targets: Model Organisms 30025.2.5 Experimental Target Identification in Humans 30025.2.6 Difference Between Target Candidates and Genuine Targets 30125.2.7 Biologicals 30125.2.8 DNA and RNA in New Therapeutic Approaches 30225.2.9 Patent Protection for Targets 30325.2.10 Compound Libraries as a Source of Drug Discovery 30425.2.11 High-Throughput Screening 30425.2.12 High-Quality Paramounts in Screening Assays 30425.2.13 Virtual Ligand Screening 30625.2.14 Activity of Drugs Described in Terms of Efficacy and Potency 30725.2.15 Chemical Optimization of Lead Structures 30725.3 Preclinical Pharmacology and Toxicology 30825.4 Clinical Development 30925.5 Clinical Testing 309Further Reading 31026 Drug Targeting and Prodrugs 311Gert Fricker26.1 Drug Targeting 31126.1.1 Passive Targeting by Exploiting Special Physiological Properties of the Target Tissue 31126.1.2 Physical Targeting 31226.1.3 Active Targeting 31326.1.4 Cellular Carrier Systems 31626.2 Prodrugs 31626.2.1 Prodrugs to Improve Drug Solubility 31626.2.2 Prodrugs to Increase Stability 31726.3 Penetration of Drugs Through Biological Membranes 31726.4 Prodrugs to Extend Duration of Effect 31826.5 Prodrugs for the Targeted Release of a Drug 31826.6 Prodrugs to Minimize Side Effects 320References 32027 Molecular Diagnostics in Medicine 323Stefan Wölfl and Reinhard Gessner27.1 Introduction 32327.2 Uses of Molecular Diagnostics 32327.2.1 Introduction 32327.2.2 Monogenic and Polygenic Diseases 32327.2.3 Individual Variability in the Genome: Forensics 32527.2.4 Individual Variability in the Genome: HLA Typing 32527.2.5 Individual Variability in the Genome: Pharmacogenomics 32527.2.6 Individual Variability in the Genome: Susceptibility to Infectious Diseases 32627.2.7 Viral Diagnosis 32627.2.8 Microbial Diagnosis and Resistance Diagnosis 32727.3 Which Molecular Variations Should be Detected 32727.3.1 Point Mutations 32727.3.2 Insertions and Deletions 32827.3.3 Nucleotide Repeats 32827.3.4 Deletion or Duplication of Genes 32827.3.5 Recombination Between Chromosomes 32927.3.6 Epigenetic Changes 32927.4 Molecular Diagnostic Methods 33027.4.1 DNA/RNA Purification 33127.4.2 Detection of Target Sequence and Known Sequence Variations 33127.4.2.1 Nucleic Acid Tests 33127.4.2.2 Quantitative PCR 33227.4.2.3 Multiplexing of Nucleic Acid Detection: Nucleic Acid Microarrays 33327.4.2.4 Production and Manufacture of Microarrays 33427.4.2.5 Applications of Fragment Length Analysis 33527.4.2.6 Minisequencing 33627.4.2.7 Determination of Unknown Mutations 33627.5 Outlook 337Further Reading 338Historic Article: “News & Views” 338Reviews 338Web Link 338Textbooks 33828 Recombinant Antibodies and Phage Display 339Gustavo Marçal Schmidt Garcia Moreira and Stefan Dübel28.1 Introduction 33928.2 Generation of Specific Recombinant Antibodies 34028.2.1 Generation of Antibody Gene Libraries 34128.2.2 Selection Systems for Recombinant Antibodies 34228.2.2.1 Transgenic Mice with Human IgG Genes 34228.2.2.2 In Vitro Selection Systems 34228.3 Production and Purification of Recombinant Antibodies 34828.4 Features and Applications of Recombinant Antibodies 34928.4.1 Advantages of Recombinant Antibodies 34928.4.2 Formats and Applications of Recombinant Antibodies 35028.4.2.1 Camelid Antibodies and VH Domains 35128.4.2.2 scFv and dsFv 35128.4.2.3 scFv–Fc Fusions, Fc Engineering, and the Addition of Constant Domains 35228.4.2.4 IgG, Fusion Proteins, and Derivatives for Therapy 35228.4.2.5 Bispecific Antibodies 35428.4.2.6 Chimeric Antigen Receptors (CARs) 35528.4.3 The Future of Therapeutic Antibodies 35528.4.4 Research and In Vitro Diagnostics 35628.4.5 Intracellular and Cell-Penetrating Antibodies 35628.5 Outlook 357Further Reading 357Textbooks 357References 35829 Genetically Modified Mice and Their Impact in Medical Research 361Rolf Sprengel and Mazahir T. Hasan29.1 Overview 36129.2 Transgenic Mice 36229.2.1 Retroviral Infection 36229.2.2 Pronuclear Injection 36329.3 Homologous Recombination: Knockout (Knock-In) Mice 36429.4 Endonuclease-Based Knockout Mice 36629.5 Endonuclease-Based Knock-In Mice 36729.6 Conditionally Regulated Gene Expression 36729.7 Gene Transfer to Subpopulations of Cells 36829.7.1 Electroporation of Mouse Embryos (Plasmid DNA) 36829.7.2 Virus-Mediated Gene Transfer (Lentivirus, rAAVs) 36929.7.3 Virus-Mediated Gene Deletion (Cre/lox) 37029.7.4 Virus-Mediated Gene Knockdown (shRNA, Antagomirs) 37029.8 Impact of Genetically Modified Mice in Biomedicine 37029.8.1 Alzheimer’s Disease 37029.8.2 Amyotrophic Lateral Sclerosis (ALS) 37029.8.3 Psychological and Cognitive Disorders 37129.8.4 Autism Spectrum Disorder (ASD) 37129.8.5 Chemogenetics, Optogenetics, and Magnetogenetics 37229.9 Outlook 372Reference 373Further Reading 37330 Plant Biotechnology 375Helke Hillebrand and Rüdiger Hell30.1 Introduction 37530.1.1 Green Genetic Engineering: A New Method Toward Traditional Goals 37530.1.2 Challenges in Plant Biotechnology 37630.2 Gene Expression Control and Genome Editing 37630.2.1 Gene Expression Control 37730.2.2 Genome Editing 37730.3 Production of Transgenic Plants 37830.3.1 Transformation Systems 37930.3.1.1 Agrobacterium as a Natural Transformation System 37930.3.1.2 Biolistic Method: Gene Gun 38130.3.1.3 Plastid Transformation 38230.3.1.4 Viral Systems 38230.4 Selection of Transformed Plant Cells 38330.4.1 Requirements for an Optimal Selection Marker System 38330.4.2 Negative Selection Marker Systems 38430.4.3 Positive Selection Marker Systems 38530.4.4 Selection Systems, Genetic Engineering Safety, and Marker-Free Plants 38530.5 Regeneration of Transgenic Plants 38730.5.1 Regeneration Procedures 38730.5.2 Composition of Regeneration Media 38730.6 Plant Analysis: Identification and Characterization of Genetically Engineered Plants 38830.6.1 DNA and RNA Verification 38830.6.2 Protein Analysis 38930.6.3 Genetic and Molecular Maps 38930.6.4 Stability of Transgenic Plants 390Further Reading 39031 Biocatalysis in the Chemical Industry 393Michael Breuer and Bernhard Hauer31.1 Introduction 39331.2 Bioconversion/Enzymatic Procedures 39531.3 Development of an Enzyme for Industrial Biocatalysis 39731.3.1 Identification of Novel Biocatalysts 39731.3.2 Improvement of Biocatalysts 39931.3.3 Production of Biocatalysts 39931.3.4 Outlook 39931.3.5 Case Study 1: Screening for New Nitrilases 40031.3.6 Case Study 2: Use of Known Enzymes for New Reactions: Lipases for the Production of Optically Active Amines and Alcohols 40031.3.7 Case Study 3: Enzyme Optimization with Rational and Evolutive Methods 40131.4 Fermentative Procedures 40231.4.1 Improvement of Fermentation Processes 40231.4.2 Classical Strain Optimization 40331.4.3 Metabolic Engineering 40431.4.4 Case Study 4: Fermentative Production of n-Butanol 40531.4.5 Case Study 5: Production of Glutamic Acid with C. glutamicum 40631.4.5.1 Molecular Mechanism of Glutamate Overproduction 40631.4.6 Case Study 6: Production of Lysine with C. glutamicum 40731.4.6.1 Molecular Mechanism of Lysine Biosynthesis 40731.4.6.2 Deregulation of the Key Enzyme Aspartate Kinase 40831.4.7 Genomic Research and Functional Genomics 40931.4.8 Case Study 7: Fermentative Penicillin Production 40931.4.9 Case Study 8: Vitamin B2 Production 40931.4.9.1 Riboflavin Biosynthesis 41031.4.9.2 Classical Strain Development 410References 410Part IV Biotechnology in Industry 41132 Industrial Application: Biotech Industry,Markets, and Opportunities 413Julia Schüler32.1 Historical Overview and Definitions of Concepts 41332.2 Areas of Industrial Application of Molecular Biotechnology 41432.2.1 Red Biotechnology 41432.2.1.1 Biopharmaceutical Drug Development 41432.2.1.2 Gene and Cell Therapy 41632.2.1.3 Tissue Engineering/Regenerative Medicine 41932.2.1.4 Pharmacogenomics and Personalized Medicine 42132.2.1.5 Molecular Diagnostic Agents 42132.2.1.6 Systems Biology 42232.2.1.7 Synthetic Biology 42232.2.2 Green Biotechnology 42232.2.2.1 Transgenic Plants 42232.2.2.2 Genomic Approaches in Green Biotechnology 42332.2.2.3 Novel Food and Functional Food 42332.2.2.4 Livestock Breeding 42332.2.3 White Biotechnology 42432.3 Status Quo of the Biotech Industry Worldwide 42432.3.1 Global Overview 42432.3.2 United States 42432.3.3 Europe 42433 Patents in the Molecular Biotechnology Industry: Legal and Ethical Issues 425David Resnik33.1 Patent Law 42533.1.1 What is a Patent? 42533.1.2 How Does One Obtain a Patent? 42633.1.3 What is the Proper Subject Matter for a Patent? 42633.1.4 Types of Patents in Pharmaceutical and Molecular Biotechnology 42733.1.5 Patent Infringement 42733.1.6 International Patent Law 42833.2 Ethical and Policy Issues in Biotechnology Patents 42833.2.1 No Patents on Nature 42833.2.2 Threats to Human Dignity 42933.2.3 Problems with Access to Technology 43033.2.4 Benefit Sharing 43233.3 Conclusions 433Acknowledgments 43334 Drug Approval in the European Union and United States 435Gary Walsh34.1 Introduction 43534.2 Regulation Within the European Union 43534.2.1 The EU Regulatory Framework 43534.2.2 The EMA and National Competent Authorities 43634.2.3 New Drug Approval Routes 43734.2.3.1 The Centralized Procedure 43734.2.3.2 Decentralized Procedure and Mutual Recognition 43834.3 Regulation in the United States 43834.3.1 CDER and CBER 43934.3.2 The Approvals Procedure 43934.4 The Advent and Regulation of Biosimilars 44034.5 International Regulatory Harmonization 441References 44235 Emergence of a Biotechnology Industry 445Claus KremoserReference 451Further Reading 45136 The 101 of Founding a Biotech Company 453Claus Kremoser and Michael Wink36.1 First Steps Toward Your Own Company 45336.2 Employees: Recruitment, Remuneration, and Participation 45637 Marketing 459Claus Kremoser and Michael Wink37.1 Introduction 45937.2 What Types of Deals Are Possible? 46037.3 What Milestone or License Fees Are Effectively Paid in a Biotech/Pharma Cooperation? 46037.4 PR and IR in Biotech Companies 461Further Reading 462Websites 462Glossary 463Index 491