内容

目次

About the Series Editors xv Part I DNA Synthesis and Genome Engineering 1 1 Competition and the Future of Reading and Writing DNA 3Robert Carlson 1.1 Productivity Improvements in Biological Technologies 3 1.2 The Origin of Moore’s Law and Its Implications for Biological Technologies 5 1.3 Lessons from Other Technologies 6 1.4 Pricing Improvements in Biological Technologies 7 1.5 Prospects for New Assembly Technologies 8 1.6 Beyond Programming Genetic Instruction Sets 10 1.7 Future Prospects 10 References 11 2 Trackable Multiplex Recombineering (TRMR) and Next–Generation Genome Design Technologies: Modifying Gene Expression in E. coli by Inserting Synthetic DNA Cassettes and Molecular Barcodes 15Emily F. Freed, Gur Pines, Carrie A. Eckert, and Ryan T. Gill 2.1 Introduction 15 2.2 Current Recombineering Techniques 16 2.2.1 Recombineering Systems 17 2.2.2 Current Model of Recombination 17 2.3 Trackable Multiplex Recombineering 19 2.3.1 TRMR and T2RMR Library Design and Construction 19 2.3.2 Experimental Procedure 23 2.3.3 Analysis of Results 24 2.4 Current Challenges 25 2.4.1 TRMR and T2RMR are Currently Not Recursive 26 2.4.2 Need for More Predictable Models 26 2.5 Complementing Technologies 27 2.5.1 MAGE 27 2.5.2 CREATE 27 2.6 Conclusions 28 Definitions 28 References 29 3 Site–Directed Genome Modification with Engineered Zinc Finger Proteins 33Lauren E. Woodard, Daniel L. Galvan, and Matthew H. Wilson 3.1 Introduction to Zinc Finger DNA–Binding Domains and Cellular Repair Mechanisms 33 3.1.1 Zinc Finger Proteins 33 3.1.2 Homologous Recombination 34 3.1.3 Non–homologous End Joining 35 3.2 Approaches for Engineering or Acquiring Zinc Finger Proteins 36 3.2.1 Modular Assembly 37 3.2.2 OPEN and CoDA Selection Systems 37 3.2.3 Purchase via Commercial Avenues 38 3.3 Genome Modification with Zinc Finger Nucleases 38 3.4 Validating Zinc Finger Nuclease–Induced Genome Alteration and Specificity 40 3.5 Methods for Delivering Engineered Zinc Finger Nucleases into Cells 41 3.6 Zinc Finger Fusions to Transposases and Recombinases 41 3.7 Conclusions 42 References 43 4 Rational Efforts to Streamline the Escherichia coli Genome 49Gabriella Balikó, Viktor Vernyik, Ildikó Karcagi, Zsuzsanna Györfy, Gábor Draskovits, Tamás Fehér, and György Pósfai 4.1 Introduction 49 4.2 The Concept of a Streamlined Chassis 50 4.3 The E. coli Genome 51 4.4 Random versus Targeted Streamlining 54 4.5 Selecting Deletion Targets 55 4.5.1 General Considerations 55 4.5.1.1 Naturally Evolved Minimal Genomes 55 4.5.1.2 Gene Essentiality Studies 55 4.5.1.3 Comparative Genomics 56 4.5.1.4 In silico Models 56 4.5.1.5 Architectural Studies 56 4.5.2 Primary Deletion Targets 57 4.5.2.1 Prophages 57 4.5.2.2 Insertion Sequences (ISs) 57 4.5.2.3 Defense Systems 57 4.5.2.4 Genes of Unknown and Exotic Functions 58 4.5.2.5 Repeat Sequences 58 4.5.2.6 Virulence Factors and Surface Structures 58 4.5.2.7 Genetic Diversity–Generating Factors 59 4.5.2.8 Redundant and Overlapping Functions 59 4.6 Targeted Deletion Techniques 59 4.6.1 General Considerations 59 4.6.2 Basic Methods and Strategies 60 4.6.2.1 Circular DNA–Based Method 60 4.6.2.2 Linear DNA–Based Method 62 4.6.2.3 Strategy for Piling Deletions 62 4.6.2.4 New Variations on Deletion Construction 63 4.7 Genome–Reducing Efforts and the Impact of Streamlining 64 4.7.1 Comparative Genomics–Based Genome Stabilization and Improvement 64 4.7.2 Genome Reduction Based on Gene Essentiality 66 4.7.3 Complex Streamlining Efforts Based on Growth Properties 67 4.7.4 Additional Genome Reduction Studies 68 4.8 Selected Research Applications of Streamlined–Genome E. coli 68 4.8.1 Testing Genome Streamlining Hypotheses 68 4.8.2 Mobile Genetic Elements, Mutations, and Evolution 69 4.8.3 Gene Function and Network Regulation 69 4.8.4 Codon Reassignment 70 4.8.5 Genome Architecture 70 4.9 Concluding Remarks, Challenges, and Future Directions 71 References 73 5 Functional Requirements in the Program and the Cell Chassis for Next–Generation Synthetic Biology 81Antoine Danchin, Agnieszka Sekowska, and Stanislas Noria 5.1 A Prerequisite to Synthetic Biology: An Engineering Definition of What Life Is 81 5.2 Functional Analysis: Master Function and Helper Functions 83 5.3 A Life–Specific Master Function: Building Up a Progeny 85 5.4 Helper Functions 86 5.4.1 Matter: Building Blocks and Structures (with Emphasis on DNA) 87 5.4.2 Energy 91 5.4.3 Managing Space 92 5.4.4 Time 95 5.4.5 Information 96 5.5 Conclusion 97 Acknowledgments 98 References 98 Part II Parts and Devices Supporting Control of Protein Expression and Activity 107 6 Constitutive and Regulated Promoters in Yeast: How to Design and Make Use of Promoters in S. cerevisiae 109Diana S. M. Ottoz and Fabian Rudolf 6.1 Introduction 109 6.2 Yeast Promoters 110 6.3 Natural Yeast Promoters 113 6.3.1 Regulated Promoters 113 6.3.2 Constitutive Promoters 115 6.4 Synthetic Yeast Promoters 116 6.4.1 Modified Natural Promoters 116 6.4.2 Synthetic Hybrid Promoters 117 6.5 Conclusions 121 Definitions 122 References 122 7 Splicing and Alternative Splicing Impact on Gene Design 131Beatrix Suess, Katrin Kemmerer, and Julia E. Weigand 7.1 The Discovery of “Split Genes” 131 7.2 Nuclear Pre–mRNA Splicing in Mammals 132 7.2.1 Introns and Exons: A Definition 132 7.2.2 The Catalytic Mechanism of Splicing 132 7.2.3 A Complex Machinery to Remove Nuclear Introns: The Spliceosome 132 7.2.4 Exon Definition 134 7.3 Splicing in Yeast 135 7.3.1 Organization and Distribution of Yeast Introns 135 7.4 Splicing without the Spliceosome 136 7.4.1 Group I and Group II Self–Splicing Introns 136 7.4.2 tRNA Splicing 137 7.5 Alternative Splicing in Mammals 137 7.5.1 Different Mechanisms of Alternative Splicing 137 7.5.2 Auxiliary Regulatory Elements 139 7.5.3 Mechanisms of Splicing Regulation 140 7.5.4 Transcription–Coupled Alternative Splicing 142 7.5.5 Alternative Splicing and Nonsense–Mediated Decay 143 7.5.6 Alternative Splicing and Disease 144 7.6 Controlled Splicing in S. cerevisiae 145 7.6.1 Alternative Splicing 145 7.6.2 Regulated Splicing 146 7.6.3 Function of Splicing in S. cerevisiae 147 7.7 Splicing Regulation by Riboswitches 147 7.7.1 Regulation of Group I Intron Splicing in Bacteria 148 7.7.2 Regulation of Alternative Splicing by Riboswitches in Eukaryotes 148 7.8 Splicing and Synthetic Biology 150 7.8.1 Impact of Introns on Gene Expression 150 7.8.2 Control of Splicing by Engineered RNA–Based Devices 151 7.9 Conclusion 153 Acknowledgments 153 Definitions 153 References 153 8 Design of Ligand–Controlled Genetic Switches Based on RNA Interference 169Shunnichi Kashida and Hirohide Saito 8.1 Utility of the RNAi Pathway for Application in Mammalian Cells 169 8.2 Development of RNAi Switches that Respond to Trigger Molecules 170 8.2.1 Small Molecule–Triggered RNAi Switches 171 8.2.2 Oligonucleotide–Triggered RNAi Switches 173 8.2.3 Protein–Triggered RNAi Switches 174 8.3 Rational Design of Functional RNAi Switches 174 8.4 Application of the RNAi Switches 175 8.5 Future Perspectives 177 Definitions 178 References 178 9 Small Molecule–Responsive RNA Switches (Bacteria): Important Element of Programming Gene Expression in Response to Environmental Signals in Bacteria 181Yohei Yokobayashi 9.1 Introduction 181 9.2 Design Strategies 181 9.2.1 Aptamers 181 9.2.2 Screening and Genetic Selection 182 9.2.3 Rational Design 183 9.3 Mechanisms 183 9.3.1 Translational Regulation 183 9.3.2 Transcriptional Regulation 184 9.4 Complex Riboswitches 185 9.5 Conclusions 185 Keywords with Definitions 185 References 186 10 Programming Gene Expression by Engineering Transcript Stability Control and Processing in Bacteria 189Jason T. Stevens and James M. Carothers 10.1 An Introduction to Transcript Control 189 10.1.1 Why Consider Transcript Control? 189 10.1.2 The RNA Degradation Process in E. coli 190 10.1.3 The Effects of Translation on Transcript Stability 192 10.1.4 Structural and Noncoding RNA–Mediated Transcript Control 193 10.1.5 Polyadenylation and Transcript Stability 195 10.2 Synthetic Control of Transcript Stability 195 10.2.1 Transcript Stability Control as a “Tuning Knob” 195 10.2.2 Secondary Structure at the 5' and 3' Ends 196 10.2.3 Noncoding RNA–Mediated 197 10.2.4 Model–Driven Transcript Stability Control for Metabolic Pathway Engineering 198 10.3 Managing Transcript Stability 201 10.3.1 Transcript Stability as a Confounding Factor 201 10.3.2 Anticipating Transcript Stability Issues 201 10.3.3 Uniformity of 5' and 3' Ends 202 10.3.4 RBS Sequestration by Riboregulators and Riboswitches 203 10.3.5 Experimentally Probing Transcript Stability 204 10.4 Potential Mechanisms for Transcript Control 205 10.4.1 Leveraging New Tools 205 10.4.2 Unused Mechanisms Found in Nature 206 10.5 Conclusions and Discussion 207 Acknowledgments 208 Definitions 208 References 209 11 Small Functional Peptides and Their Application in Superfunctionalizing Proteins 217Sonja Billerbeck 11.1 Introduction 217 11.2 Permissive Sites and Their Identification in a Protein 218 11.3 Functional Peptides 220 11.3.1 Functional Peptides that Act as Binders 220 11.3.2 Peptide Motifs that are Recognized by Labeling Enzymes 221 11.3.3 Peptides as Protease Cleavage Sites 222 11.3.4 Reactive Peptides 223 11.3.5 Pharmaceutically Relevant Peptides: Peptide Epitopes, Sugar Epitope Mimics, and Antimicrobial Peptides 223 11.3.5.1 Peptide Epitopes 224 11.3.5.2 Peptide Mimotopes 224 11.3.5.3 Antimicrobial Peptides 225 11.4 Conclusions 227 Definitions 228 Abbreviations 228 Acknowledgment 229 References 229 Part III Parts and Devices Supporting Spatial Engineering 237 12 Metabolic Channeling Using DNA as a Scaffold 239Mojca Benèina, Jerneja Mori, Rok Gaber, and Roman Jerala 12.1 Introduction 239 12.2 Biosynthetic Applications of DNA Scaffold 242 12.2.1 l–Threonine 242 12.2.2 trans–Resveratrol 245 12.2.3 1,2–Propanediol 246 12.2.4 Mevalonate 246 12.3 Design of DNA–Binding Proteins and Target Sites 247 12.3.1 Zinc Finger Domains 248 12.3.2 TAL–DNA Binding Domains 249 12.3.3 Other DNA–Binding Proteins 250 12.4 DNA Program 250 12.4.1 Spacers between DNA–Target Sites 250 12.4.2 Number of DNA Scaffold Repeats 252 12.4.3 DNA–Target Site Arrangement 253 12.5 Applications of DNA–Guided Programming 254 Definitions 255 References 256 13 Synthetic RNA Scaffolds for Spatial Engineering in Cells 261Gairik Sachdeva, Cameron Myhrvold, Peng Yin, and Pamela A. Silver 13.1 Introduction 261 13.2 Structural Roles of Natural RNA 261 13.2.1 RNA as a Natural Catalyst 262 13.2.2 RNA Scaffolds in Nature 263 13.3 Design Principles for RNA Are Well Understood 263 13.3.1 RNA Secondary Structure is Predictable 264 13.3.2 RNA can Self–Assemble into Structures 265 13.3.3 Dynamic RNAs can be Rationally Designed 265 13.3.4 RNA can be Selected in vitro to Enhance Its Function 266 13.4 Applications of Designed RNA Scaffolds 266 13.4.1 Tools for RNA Research 266 13.4.2 Localizing Metabolic Enzymes on RNA 267 13.4.3 Packaging Therapeutics on RNA Scaffolds 269 13.4.4 Recombinant RNA Technology 269 13.5 Conclusion 270 13.5.1 New Applications 270 13.5.2 Technological Advances 270 Definitions 271 References 271 14 Sequestered: Design and Construction of Synthetic Organelles 279Thawatchai Chaijarasphong and David F. Savage 14.1 Introduction 279 14.2 On Organelles 281 14.3 Protein–Based Organelles 283 14.3.1 Bacterial Microcompartments 283 14.3.1.1 Targeting 285 14.3.1.2 Permeability 287 14.3.1.3 Chemical Environment 288 14.3.1.4 Biogenesis 289 14.3.2 Alternative Protein Organelles: A Minimal System 290 14.4 Lipid–Based Organelles 292 14.4.1 Repurposing Existing Organelles 293 14.4.1.1 The Mitochondrion 293 14.4.1.2 The Vacuole 294 14.5 De novo Organelle Construction and Future Directions 295 Acknowledgments 297 References 297 Part IV Early Applications of Synthetic Biology: Pathways, Therapies, and Cell–Free Synthesis 307 15 Cell–Free Protein Synthesis: An Emerging Technology for Understanding, Harnessing, and Expanding the Capabilities of Biological Systems 309Jennifer A. Schoborg and Michael C. Jewett 15.1 Introduction 309 15.2 Background/Current Status 311 15.2.1 Platforms 311 15.2.1.1 Prokaryotic Platforms 311 15.2.1.2 Eukaryotic Platforms 312 15.2.2 Trends 314 15.3 Products 316 15.3.1 Noncanonical Amino Acids 316 15.3.2 Glycosylation 316 15.3.3 Antibodies 318 15.3.4 Membrane Proteins 318 15.4 High–Throughput Applications 320 15.4.1 Protein Production and Screening 320 15.4.2 Genetic Circuit Optimization 321 15.5 Future of the Field 321 Definitions 322 Acknowledgments 322 References 323 16 Applying Advanced DNA Assembly Methods to Generate Pathway Libraries 331Dawn T. Eriksen, Ran Chao, and Huimin Zhao 16.1 Introduction 331 16.2 Advanced DNA Assembly Methods 333 16.3 Generation of Pathway Libraries 334 16.3.1 In vitro Assembly Methods 335 16.3.2 In vivo Assembly Methods 339 16.3.2.1 In vivo Chromosomal Integration 339 16.3.2.2 In vivo Plasmid Assembly and One–Step Optimization Libraries 340 16.3.2.3 In vivo Plasmid Assembly and Iterative Multi–step Optimization Libraries 341 16.4 Conclusions and Prospects 343 Definitions 343 References 344 17 Synthetic Biology in Immunotherapy and Stem Cell Therapy Engineering 349Patrick Ho and Yvonne Y. Chen 17.1 The Need for a New Therapeutic Paradigm 349 17.2 Rationale for Cellular Therapies 350 17.3 Synthetic Biology Approaches to Cellular Immunotherapy Engineering 351 17.3.1 CAR Engineering for Adoptive T–Cell Therapy 352 17.3.2 Genetic Engineering to Enhance T–Cell Therapeutic Function 357 17.3.3 Generating Safer T–Cell Therapeutics with Synthetic Biology 359 17.4 Challenges and Future Outlook 362 Acknowledgment 364 Definitions 364 References 365 Part V Societal Ramifications of Synthetic Biology 373 18 Synthetic Biology: From Genetic Engineering 2.0 to Responsible Research and Innovation 375Lei Pei and Markus Schmidt 18.1 Introduction 375 18.2 Public Perception of the Nascent Field of Synthetic Biology 376 18.2.1 Perception of Synthetic Biology in the United States 377 18.2.2 Perception of Synthetic Biology in Europe 379 18.2.2.1 European Union 379 18.2.2.2 Austria 379 18.2.2.3 Germany 381 18.2.2.4 Netherlands 382 18.2.2.5 United Kingdom 383 18.2.3 Opinions from Concerned Civil Society Groups 384 18.3 Frames and Comparators 384 18.3.1 Genetic Engineering: Technology as Conflict 386 18.3.2 Nanotechnology: Technology as Progress 387 18.3.3 Information Technology: Technology as Gadget 387 18.3.4 SB: Which Debate to Come? 388 18.4 Toward Responsible Research and Innovation (RRI) in Synthetic Biology 389 18.4.1 Engagement of All Societal Actors – Researchers, Industry, Policy Makers, and Civil Society – and Their Joint Participation in the Research and Innovation 390 18.4.2 Gender Equality 391 18.4.3 Science Education 392 18.4.4 Open Access 392 18.4.5 Ethics 394 18.4.6 Governance 395 18.5 Conclusion 396 Acknowledgments 397 References 397 Index 403