Synthetic Biology, Part A (eBook)

Front cover 
1 
Synthetic Biology, Part A: Methods for Part/Device Characterization and Chassis Engineering 4
Copyright 5
Contents 6
Contributors 14
Preface 22
Methods in Enzymology 24
Section One: Measuring and Engineering Central Dogma Processes 56
Chapter 1: Sequence-Specificity and Energy Landscapes of DNA-Binding Molecules 58
1. Introduction 59
2. Array-Based Cognate Sequence Identification 61
3. Solution-Based Cognate Sequence Identification 68
4. Data Analysis and Visualization of Specificity, Binding Energy, and Genomic-Association Landscapes 73
References 82
Chapter 2: Promoter Reliability in Modular Transcriptional Networks 86
1. Results 88
2. Conclusion 99
3. Methods 100
Acknowledgments 102
References 102
Chapter 3: The Analysis of ChIP-Seq Data 106
1. Introduction 107
2. Planning of ChIP-Seq Experiments 108
3. Processing and Analyzing ChIP-Seq Datasets 113
4. Discussion 123
Acknowledgments 125
References 126
Chapter 4: Using DNA Microarrays to Assay Part Function 130
1. Introduction 131
2. Different Microarray Platforms 132
3. Experimental Design 136
4. Experimental Variation 137
5. Sample Preparation 140
6. Microarray Preprocessing 148
7. Clustering 154
8. Differential Expression Analysis 159
9. Data Analysis: Understanding the Perturbation 161
10. Closing Remarks 163
References 164
Chapter 5: Orthogonal Gene Expression in Escherichia coli 170
1. Introduction 171
2. High-Throughput Screening for Orthogonal T7 Promoter O-rbs System 174
3. Integration of Orthogonal Pairs to Synthesize Transcription-Translation FFL 177
4. Engineering the FFL Delay via the Discovery of a Minimal O-rRNA 178
5. Discussion 181
6. Material and Methods 184
Acknowledgments 187
References 187
Chapter 6: Directed Evolution of Promoters and Tandem Gene Arrays for Customizing RNA Synthesis Rates and Regulation 190
1. Introduction 191
2. Promoter Modification by Error-Prone PCR 193
3. Generating Stable Tandem Gene Arrays for Controlling RNA Synthesis Rate 205
4. Concluding Remarks 208
References 209
Section Two: Device and System Design, Optimization, and Debugging 212
Chapter 7: Design and Connection of Robust Genetic Circuits 214
1. Introduction 215
2. Sources of Failure 216
3. Robustness Principles and Examples in Natural Systems 218
4. Methods for Obtaining Robust Synthetic Circuits 220
5. Robustness Trade-Offs 236
6. Conclusion 237
References 237
Chapter 8: Engineering RNAi Circuits 242
1. Introduction 243
2. Constructing a Computational Logic Core for the RNAi-Based DNF Circuit 244
3. Constructing a Computational Logic Core for the RNAi-Based CNF Circuit 255
4. Transition from siRNA to miRNA 257
Acknowledgments 259
References 259
Chapter 9: From SELEX to Cell... 262
1. Introduction 262
2. General Precautions 263
3. In Vitro Selection 263
4. In Vivo Selection 267
References 274
Chapter 10: Using Noisy Gene Expression Mediated by Engineered Adenovirus to Probe Signaling Dynamics in Mammalian Cells 276
1. Introduction 277
2. Design and Construction 279
3. Measurement 285
4. Broader Applications 289
References 289
Chapter 11: De novo Design and Construction of an Inducible Gene Expression System in Mammalian Cells 294
1. Introduction 295
2. Selection of a Conditional DNA-Binding Protein 298
3. Establishment of the Inducible Expression System 299
4. Optimization of the Expression System 303
5. Summary 305
Acknowledgments 306
References 306
Chapter 12: BioBuilding... 310
1. Introduction 311
2. Eau d'coli 312
3. "Eau That Smell" Teaching Lab Using the MIT iGEM Team's Eau d'coli Cells 314
4. Teaching Labs Modified for Resource-Stretched Settings 322
5. Summary 324
Acknowledgments 325
References 325
Section Three: Device Measurement, Optimization, and Debugging 328
Chapter 13: Use of Fluorescence Microscopy to Analyze Genetic Circuit Dynamics 330
1. Fluorescent Reporters 331
2. Constructing and Using Genetic Fluorescent Reporters 332
3. Fluorescent Time-Lapse Microscopy 336
4. Measuring and Interpreting Dynamics 339
5. Applications for Measurement of Circuit Dynamics 341
References 347
Chapter 14: Microfluidics for Synthetic Biology 350
1. Part I: Introduction 351
2. Part II: Fabrication 393
3. Part III: Experiments 413
Appendix 424
Acknowledgments 426
References 426
Chapter 15: Plate-Based Assays for Light-Regulated Gene Expression Systems 428
1. Bacterial Photography Protocol 429
2. Bacterial Edge Detection Protocol 434
3. Setting up a Projector-Incubator 436
4. The beta-Galactosidase/S-Gal Reporter System 439
5. Quantifying Signal Intensity on the Plates 440
6. Microscopic Imaging of Agarose Slabs 440
7. Properties of Relevant Strains 441
8. Properties of Relevant Plasmids 442
References 445
Chapter 16: Spatiotemporal Control of Small GTPases with Light Using the LOV Domain 448
1. Introduction 449
2. The LOV Domain as a Tool for Protein Caging 450
3. Design and Structure Optimization of PA-Rac 450
4. Activation of PA-Rac in Living Cells 452
5. Application of PA-Rac in Drosophila Ovarian Border Cell Migration 455
References 462
Chapter 17: Light Control of Plasma Membrane Recruitment Using the Phy-PIF System 464
1. Introduction 465
2. Light-Controlled Phy-PIF Interaction 466
3. Genetic Constructs Encoding Phy and PIF Components 467
4. Purification of PCB from Spirulina 470
5. Cell Culture Preparation for Phy-PIF Translocation 473
6. Imaging PIF Translocation Using Spinning Disk Confocal Microscopy 474
Acknowledgments 476
References 476
Chapter 18: Synthetic Physiology... 480
1. Introduction 481
2. Molecular Design and Construction 484
3. Transduction of Microbial Opsins into Cells for Heterologous Expression 487
4. Physiological Assays 490
5. Conclusion 493
Acknowledgments 494
References 494
Section Four: Devices for Metabolic Engineering 500
Chapter 19: Metabolic Pathway Flux Enhancement by Synthetic Protein Scaffolding 502
1. Introduction 503
2. Method-How to Build Modular Protein Scaffolded Systems for Metabolic Engineering Applications 509
3. Systems that May Benefit from Scaffolding 520
4. Concluding Remarks 520
Acknowledgments 521
References 521
Chapter 20: A Synthetic Iterative Pathway for Ketoacid Elongation 524
1. Introduction 525
2. Natural Pathways Involving Ketoacid Chain Elongations Catalyzed by the LeuABCD-Dependent Mechanisms 526
3. IPMS and Similar Enzymes 528
4. Expansion to Nonnatural Pathways 530
5. Transfer of Citramalate Pathway to E. coli for Ketoacid Chain Elongation 533
6. Conclusion Remarks 535
References 535
Section Five: Expanding Chassis 538
Chapter 21: Synthetic Biology in Streptomyces Bacteria 540
1. Synthetic Biology for Novel Compound Discovery in Streptomyces 541
2. Practical Considerations for Synthetic Biology in Streptomyces 543
3. Iterative Reengineering of Secondary Metabolite Gene Clusters 544
4. The Molecular Toolbox for Streptomyces Synthetic Biology 546
5. Transcriptional Control 547
6. Translational Control 549
7. Vectors 549
Acknowledgments 552
References 552
Chapter 22: Methods for Engineering Sulfate Reducing Bacteria of the Genus Desulfovibrio 558
1. Introduction 559
2. Chromosomal Modifications Through Homologous Recombination 560
3. Culturing Conditions and Antibiotic Selection 562
4. DNA Transformation 565
5. Screening Colonies for Proper Integration 568
6. Complementing Gene Deletions 569
7 Concluding Remarks 570
Acknowledgments 571
References 571
Chapter 23: Modification of the Genome of Rhodobacter sphaeroides and Construction of Synthetic Operons 574
1. Introduction 575
2. Gene Disruption and Deletion 577
3. Construction of Synthetic Operons 582
4. Future Directions 587
References 588
Chapter 24: Synthetic Biology in Cyanobacteria... 594
1. Introduction 595
2. Cyanobacterial Chassis 597
3. Biological Parts in Cyanobacteria 599
4. Genetic Engineering of Cyanobacteria 605
5. Molecular Analysis of Cyanobacteria 617
6. Conclusion and Outlook 626
Acknowledgments 627
References 627
Chapter 25: Developing a Synthetic Signal Transduction System in Plants 636
1. Introduction 637
2. Foundation for Developing a Molecular Testing Platform for HK Systems 641
3. Technical Considerations in Developing a Eukaryotic Synthetic Signal Transduction System Based on Bacterial TCS Components. 644
4. A Partial Synthetic Signal Transduction System Using Cytokinin Input 647
5. A Eukaryotic Synthetic Signal Transduction Pathway 648
6. Conclusions 650
7. Protocols 652
Acknowledgments 654
References 654
Chapter 26: Lentiviral Vectors to Study Stochastic Noise in Gene Expression 658
1. Introduction 659
2. The Lentiviral-Vector Approach 660
3. Production of Lentiviral Vectors and Transduced Cell Lines 664
4. Procedure for Constructing a CV2 Versus Mean Plot 671
5. Inferring Promoter Regulatory Architecture from CV2 Versus Mean Analysis 671
6. Conclusion 675
Acknowledgments 675
References 675
Author Index 678
Subject Index 706
Colour Plate 718