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 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...