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 Moores 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, andGyörgy 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 Bacteria189Jason 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 Beneina, 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
