Title Details

Sang Yup Lee is Distinguished Professor at the Department of Chemical and Biomolecular Engineering at the Korea Advanced Institute of Science and Technology (KAIST).

Jens Nielsen is Professor and Director to Chalmers University of Technology, Sweden. He has received numerous Danish and international awards including the Nature Mentor Award.

Professor Gregory Stephanopoulos is the W. H. Dow Professor of Chemical Engineering at the Massachusetts Institute of Technology and Director of the MIT Metabolic Engineering Laboratory. 

About the Series Editors xv

Part I DNA Synthesis and Genome Engineering 1

1 Competition and the Future of Reading and Writing DNA 3
Robert 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 15
Emily 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 33
Lauren 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 49
Gabriella 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 81
Antoine 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 109
Diana 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 131
Beatrix 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 169
Shunnichi 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 181
Yohei 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 Bacteria189
Jason 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 217
Sonja 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 239
Mojca 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 261
Gairik 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 279
Thawatchai 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 309
Jennifer 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 331
Dawn 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 349
Patrick 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 375
Lei 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