Synthetic Biology

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 xvPart I DNA Synthesis and Genome Engineering 11 Competition and the Future of Reading and Writing DNA 3Robert Carlson1.1 Productivity Improvements in Biological Technologies 31.2 The Origin of Moore’s Law and Its Implications for Biological Technologies 51.3 Lessons from Other Technologies 61.4 Pricing Improvements in Biological Technologies 71.5 Prospects for New Assembly Technologies 81.6 Beyond Programming Genetic Instruction Sets 101.7 Future Prospects 10References 112 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. Gill2.1 Introduction 152.2 Current Recombineering Techniques 162.2.1 Recombineering Systems 172.2.2 Current Model of Recombination 172.3 Trackable Multiplex Recombineering 192.3.1 TRMR and T2RMR Library Design and Construction 192.3.2 Experimental Procedure 232.3.3 Analysis of Results 242.4 Current Challenges 252.4.1 TRMR and T2RMR are Currently Not Recursive 262.4.2 Need for More Predictable Models 262.5 Complementing Technologies 272.5.1 MAGE 272.5.2 CREATE 272.6 Conclusions 28Definitions 28References 293 Site-Directed Genome Modification with Engineered Zinc Finger Proteins 33Lauren E. Woodard, Daniel L. Galvan, and Matthew H. Wilson3.1 Introduction to Zinc Finger DNA-Binding Domains and Cellular Repair Mechanisms 333.1.1 Zinc Finger Proteins 333.1.2 Homologous Recombination 343.1.3 Non-homologous End Joining 353.2 Approaches for Engineering or Acquiring Zinc Finger Proteins 363.2.1 Modular Assembly 373.2.2 OPEN and CoDA Selection Systems 373.2.3 Purchase via Commercial Avenues 383.3 Genome Modification with Zinc Finger Nucleases 383.4 Validating Zinc Finger Nuclease-Induced Genome Alteration and Specificity 403.5 Methods for Delivering Engineered Zinc Finger Nucleases into Cells 413.6 Zinc Finger Fusions to Transposases and Recombinases 413.7 Conclusions 42References 434 Rational Efforts to Streamline the Escherichia coli Genome 49Gabriella Balikó, Viktor Vernyik, Ildikó Karcagi, Zsuzsanna Györfy, Gábor Draskovits, Tamás Fehér, and György Pósfai4.1 Introduction 494.2 The Concept of a Streamlined Chassis 504.3 The E. coli Genome 514.4 Random versus Targeted Streamlining 544.5 Selecting Deletion Targets 554.5.1 General Considerations 554.5.1.1 Naturally Evolved Minimal Genomes 554.5.1.2 Gene Essentiality Studies 554.5.1.3 Comparative Genomics 564.5.1.4 In silico Models 564.5.1.5 Architectural Studies 564.5.2 Primary Deletion Targets 574.5.2.1 Prophages 574.5.2.2 Insertion Sequences (ISs) 574.5.2.3 Defense Systems 574.5.2.4 Genes of Unknown and Exotic Functions 584.5.2.5 Repeat Sequences 584.5.2.6 Virulence Factors and Surface Structures 584.5.2.7 Genetic Diversity-Generating Factors 594.5.2.8 Redundant and Overlapping Functions 594.6 Targeted Deletion Techniques 594.6.1 General Considerations 594.6.2 Basic Methods and Strategies 604.6.2.1 Circular DNA-Based Method 604.6.2.2 Linear DNA-Based Method 624.6.2.3 Strategy for Piling Deletions 624.6.2.4 New Variations on Deletion Construction 634.7 Genome-Reducing Efforts and the Impact of Streamlining 644.7.1 Comparative Genomics-Based Genome Stabilization and Improvement 644.7.2 Genome Reduction Based on Gene Essentiality 664.7.3 Complex Streamlining Efforts Based on Growth Properties 674.7.4 Additional Genome Reduction Studies 684.8 Selected Research Applications of Streamlined-Genome E. coli 684.8.1 Testing Genome Streamlining Hypotheses 684.8.2 Mobile Genetic Elements, Mutations, and Evolution 694.8.3 Gene Function and Network Regulation 694.8.4 Codon Reassignment 704.8.5 Genome Architecture 704.9 Concluding Remarks, Challenges, and Future Directions 71References 735 Functional Requirements in the Program and the Cell Chassis for Next-Generation Synthetic Biology 81Antoine Danchin, Agnieszka Sekowska, and Stanislas Noria5.1 A Prerequisite to Synthetic Biology: An Engineering Definition of What Life Is 815.2 Functional Analysis: Master Function and Helper Functions 835.3 A Life-Specific Master Function: Building Up a Progeny 855.4 Helper Functions 865.4.1 Matter: Building Blocks and Structures (with Emphasis on DNA) 875.4.2 Energy 915.4.3 Managing Space 925.4.4 Time 955.4.5 Information 965.5 Conclusion 97Acknowledgments 98References 98Part II Parts and Devices Supporting Control of Protein Expression and Activity 1076 Constitutive and Regulated Promoters in Yeast: How to Design and Make Use of Promoters in S. cerevisiae 109Diana S. M. Ottoz and Fabian Rudolf6.1 Introduction 1096.2 Yeast Promoters 1106.3 Natural Yeast Promoters 1136.3.1 Regulated Promoters 1136.3.2 Constitutive Promoters 1156.4 Synthetic Yeast Promoters 1166.4.1 Modified Natural Promoters 1166.4.2 Synthetic Hybrid Promoters 1176.5 Conclusions 121Definitions 122References 1227 Splicing and Alternative Splicing Impact on Gene Design 131Beatrix Suess, Katrin Kemmerer, and Julia E. Weigand7.1 The Discovery of “Split Genes” 1317.2 Nuclear Pre-mRNA Splicing in Mammals 1327.2.1 Introns and Exons: A Definition 1327.2.2 The Catalytic Mechanism of Splicing 1327.2.3 A Complex Machinery to Remove Nuclear Introns: The Spliceosome 1327.2.4 Exon Definition 1347.3 Splicing in Yeast 1357.3.1 Organization and Distribution of Yeast Introns 1357.4 Splicing without the Spliceosome 1367.4.1 Group I and Group II Self-Splicing Introns 1367.4.2 tRNA Splicing 1377.5 Alternative Splicing in Mammals 1377.5.1 Different Mechanisms of Alternative Splicing 1377.5.2 Auxiliary Regulatory Elements 1397.5.3 Mechanisms of Splicing Regulation 1407.5.4 Transcription-Coupled Alternative Splicing 1427.5.5 Alternative Splicing and Nonsense-Mediated Decay 1437.5.6 Alternative Splicing and Disease 1447.6 Controlled Splicing in S. cerevisiae 1457.6.1 Alternative Splicing 1457.6.2 Regulated Splicing 1467.6.3 Function of Splicing in S. cerevisiae 1477.7 Splicing Regulation by Riboswitches 1477.7.1 Regulation of Group I Intron Splicing in Bacteria 1487.7.2 Regulation of Alternative Splicing by Riboswitches in Eukaryotes 1487.8 Splicing and Synthetic Biology 1507.8.1 Impact of Introns on Gene Expression 1507.8.2 Control of Splicing by Engineered RNA-Based Devices 1517.9 Conclusion 153Acknowledgments 153Definitions 153References 1538 Design of Ligand-Controlled Genetic Switches Based on RNA Interference 169Shunnichi Kashida and Hirohide Saito8.1 Utility of the RNAi Pathway for Application in Mammalian Cells 1698.2 Development of RNAi Switches that Respond to Trigger Molecules 1708.2.1 Small Molecule-Triggered RNAi Switches 1718.2.2 Oligonucleotide-Triggered RNAi Switches 1738.2.3 Protein-Triggered RNAi Switches 1748.3 Rational Design of Functional RNAi Switches 1748.4 Application of the RNAi Switches 1758.5 Future Perspectives 177Definitions 178References 1789 Small Molecule-Responsive RNA Switches (Bacteria): Important Element of Programming Gene Expression in Response to Environmental Signals in Bacteria 181Yohei Yokobayashi9.1 Introduction 1819.2 Design Strategies 1819.2.1 Aptamers 1819.2.2 Screening and Genetic Selection 1829.2.3 Rational Design 1839.3 Mechanisms 1839.3.1 Translational Regulation 1839.3.2 Transcriptional Regulation 1849.4 Complex Riboswitches 1859.5 Conclusions 185Keywords with Definitions 185References 18610 Programming Gene Expression by Engineering Transcript Stability Control and Processing in Bacteria 189Jason T. Stevens and James M. Carothers10.1 An Introduction to Transcript Control 18910.1.1 Why Consider Transcript Control? 18910.1.2 The RNA Degradation Process in E. coli 19010.1.3 The Effects of Translation on Transcript Stability 19210.1.4 Structural and Noncoding RNA-Mediated Transcript Control 19310.1.5 Polyadenylation and Transcript Stability 19510.2 Synthetic Control of Transcript Stability 19510.2.1 Transcript Stability Control as a “Tuning Knob” 19510.2.2 Secondary Structure at the 5′ and 3′ Ends 19610.2.3 Noncoding RNA-Mediated 19710.2.4 Model-Driven Transcript Stability Control for Metabolic Pathway Engineering 19810.3 Managing Transcript Stability 20110.3.1 Transcript Stability as a Confounding Factor 20110.3.2 Anticipating Transcript Stability Issues 20110.3.3 Uniformity of 5′ and 3′ Ends 20210.3.4 RBS Sequestration by Riboregulators and Riboswitches 20310.3.5 Experimentally Probing Transcript Stability 20410.4 Potential Mechanisms for Transcript Control 20510.4.1 Leveraging New Tools 20510.4.2 Unused Mechanisms Found in Nature 20610.5 Conclusions and Discussion 207Acknowledgments 208Definitions 208References 20911 Small Functional Peptides and Their Application in Superfunctionalizing Proteins 217Sonja Billerbeck11.1 Introduction 21711.2 Permissive Sites and Their Identification in a Protein 21811.3 Functional Peptides 22011.3.1 Functional Peptides that Act as Binders 22011.3.2 Peptide Motifs that are Recognized by Labeling Enzymes 22111.3.3 Peptides as Protease Cleavage Sites 22211.3.4 Reactive Peptides 22311.3.5 Pharmaceutically Relevant Peptides: Peptide Epitopes, Sugar Epitope Mimics, and Antimicrobial Peptides 22311.3.5.1 Peptide Epitopes 22411.3.5.2 Peptide Mimotopes 22411.3.5.3 Antimicrobial Peptides 22511.4 Conclusions 227Definitions 228Abbreviations 228Acknowledgment 229References 229Part III Parts and Devices Supporting Spatial Engineering 23712 Metabolic Channeling Using DNA as a Scaffold 239Mojca Benèina, Jerneja Mori, Rok Gaber, and Roman Jerala12.1 Introduction 23912.2 Biosynthetic Applications of DNA Scaffold 24212.2.1 l-Threonine 24212.2.2 trans-Resveratrol 24512.2.3 1,2-Propanediol 24612.2.4 Mevalonate 24612.3 Design of DNA-Binding Proteins and Target Sites 24712.3.1 Zinc Finger Domains 24812.3.2 TAL-DNA Binding Domains 24912.3.3 Other DNA-Binding Proteins 25012.4 DNA Program 25012.4.1 Spacers between DNA-Target Sites 25012.4.2 Number of DNA Scaffold Repeats 25212.4.3 DNA-Target Site Arrangement 25312.5 Applications of DNA-Guided Programming 254Definitions 255References 25613 Synthetic RNA Scaffolds for Spatial Engineering in Cells 261Gairik Sachdeva, Cameron Myhrvold, Peng Yin, and Pamela A. Silver13.1 Introduction 26113.2 Structural Roles of Natural RNA 26113.2.1 RNA as a Natural Catalyst 26213.2.2 RNA Scaffolds in Nature 26313.3 Design Principles for RNA Are Well Understood 26313.3.1 RNA Secondary Structure is Predictable 26413.3.2 RNA can Self-Assemble into Structures 26513.3.3 Dynamic RNAs can be Rationally Designed 26513.3.4 RNA can be Selected in vitro to Enhance Its Function 26613.4 Applications of Designed RNA Scaffolds 26613.4.1 Tools for RNA Research 26613.4.2 Localizing Metabolic Enzymes on RNA 26713.4.3 Packaging Therapeutics on RNA Scaffolds 26913.4.4 Recombinant RNA Technology 26913.5 Conclusion 27013.5.1 New Applications 27013.5.2 Technological Advances 270Definitions 271References 27114 Sequestered: Design and Construction of Synthetic Organelles 279Thawatchai Chaijarasphong and David F. Savage14.1 Introduction 27914.2 On Organelles 28114.3 Protein-Based Organelles 28314.3.1 Bacterial Microcompartments 28314.3.1.1 Targeting 28514.3.1.2 Permeability 28714.3.1.3 Chemical Environment 28814.3.1.4 Biogenesis 28914.3.2 Alternative Protein Organelles: A Minimal System 29014.4 Lipid-Based Organelles 29214.4.1 Repurposing Existing Organelles 29314.4.1.1 The Mitochondrion 29314.4.1.2 The Vacuole 29414.5 De novo Organelle Construction and Future Directions 295Acknowledgments 297References 297Part IV Early Applications of Synthetic Biology: Pathways, Therapies, and Cell-Free Synthesis 30715 Cell-Free Protein Synthesis: An Emerging Technology for Understanding, Harnessing, and Expanding the Capabilities of Biological Systems 309Jennifer A. Schoborg and Michael C. Jewett15.1 Introduction 30915.2 Background/Current Status 31115.2.1 Platforms 31115.2.1.1 Prokaryotic Platforms 31115.2.1.2 Eukaryotic Platforms 31215.2.2 Trends 31415.3 Products 31615.3.1 Noncanonical Amino Acids 31615.3.2 Glycosylation 31615.3.3 Antibodies 31815.3.4 Membrane Proteins 31815.4 High-Throughput Applications 32015.4.1 Protein Production and Screening 32015.4.2 Genetic Circuit Optimization 32115.5 Future of the Field 321Definitions 322Acknowledgments 322References 32316 Applying Advanced DNA Assembly Methods to Generate Pathway Libraries 331Dawn T. Eriksen, Ran Chao, and Huimin Zhao16.1 Introduction 33116.2 Advanced DNA Assembly Methods 33316.3 Generation of Pathway Libraries 33416.3.1 In vitro Assembly Methods 33516.3.2 In vivo Assembly Methods 33916.3.2.1 In vivo Chromosomal Integration 33916.3.2.2 In vivo Plasmid Assembly and One-Step Optimization Libraries 34016.3.2.3 In vivo Plasmid Assembly and Iterative Multi-step Optimization Libraries 34116.4 Conclusions and Prospects 343Definitions 343References 34417 Synthetic Biology in Immunotherapy and Stem Cell Therapy Engineering 349Patrick Ho and Yvonne Y. Chen17.1 The Need for a New Therapeutic Paradigm 34917.2 Rationale for Cellular Therapies 35017.3 Synthetic Biology Approaches to Cellular Immunotherapy Engineering 35117.3.1 CAR Engineering for Adoptive T-Cell Therapy 35217.3.2 Genetic Engineering to Enhance T-Cell Therapeutic Function 35717.3.3 Generating Safer T-Cell Therapeutics with Synthetic Biology 35917.4 Challenges and Future Outlook 362Acknowledgment 364Definitions 364References 365Part V Societal Ramifications of Synthetic Biology 37318 Synthetic Biology: From Genetic Engineering 2.0 to Responsible Research and Innovation 375Lei Pei and Markus Schmidt18.1 Introduction 37518.2 Public Perception of the Nascent Field of Synthetic Biology 37618.2.1 Perception of Synthetic Biology in the United States 37718.2.2 Perception of Synthetic Biology in Europe 37918.2.2.1 European Union 37918.2.2.2 Austria 37918.2.2.3 Germany 38118.2.2.4 Netherlands 38218.2.2.5 United Kingdom 38318.2.3 Opinions from Concerned Civil Society Groups 38418.3 Frames and Comparators 38418.3.1 Genetic Engineering: Technology as Conflict 38618.3.2 Nanotechnology: Technology as Progress 38718.3.3 Information Technology: Technology as Gadget 38718.3.4 SB: Which Debate to Come? 38818.4 Toward Responsible Research and Innovation (RRI) in Synthetic Biology 38918.4.1 Engagement of All Societal Actors – Researchers, Industry, Policy Makers, and Civil Society – and Their Joint Participation in the Research and Innovation 39018.4.2 Gender Equality 39118.4.3 Science Education 39218.4.4 Open Access 39218.4.5 Ethics 39418.4.6 Governance 39518.5 Conclusion 396Acknowledgments 397References 397Index 403