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