Voigt Synthetic Biology, Part A

Methods for Part/Device Characterization and Chassis Engineering
1. Auflage 2011
ISBN: 978-0-12-385076-8
Verlag: Elsevier Science & Techn.
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, Band Volume 497, 714 Seiten, Web PDF

Reihe: Methods in Enzymology

Synthetic biology encompasses a variety of different approaches, methodologies and disciplines, and many different definitions exist. This Volume of Methods in Enzymology has been split into 2 Parts and covers topics such as Measuring and Engineering Central Dogma Processes, Mathematical and Computational Methods and Next-Generation DNA Assembly and Manipulation. - Encompasses a variety of different approaches, methodologies and disciplines - Split into 2 parts and covers topics such as measuring and engineering central dogma processes, mathematical and computational methods and next-generation DNA assembly and manipulation

Autoren/Hrsg.

Voigt, Chris

Inhaltsverzeichnis

1;Front cover
;1
2;Synthetic Biology, Part A: Methods for Part/Device Characterization and Chassis Engineering;4
3;Copyright;5
4;Contents;6
5;Contributors;14
6;Preface;22
7;Methods in Enzymology;24
8;Section One: Measuring and Engineering Central Dogma Processes;56
8.1;Chapter 1: Sequence-Specificity and Energy Landscapes of DNA-Binding Molecules;58
8.1.1;1. Introduction;59
8.1.2;2. Array-Based Cognate Sequence Identification;61
8.1.3;3. Solution-Based Cognate Sequence Identification;68
8.1.4;4. Data Analysis and Visualization of Specificity, Binding Energy, and Genomic-Association Landscapes;73
8.1.5;References;82
8.2;Chapter 2: Promoter Reliability in Modular Transcriptional Networks;86
8.2.1;1. Results;88
8.2.2;2. Conclusion;99
8.2.3;3. Methods;100
8.2.4;Acknowledgments;102
8.2.5;References;102
8.3;Chapter 3: The Analysis of ChIP-Seq Data;106
8.3.1;1. Introduction;107
8.3.2;2. Planning of ChIP-Seq Experiments;108
8.3.3;3. Processing and Analyzing ChIP-Seq Datasets;113
8.3.4;4. Discussion;123
8.3.5;Acknowledgments;125
8.3.6;References;126
8.4;Chapter 4: Using DNA Microarrays to Assay Part Function;130
8.4.1;1. Introduction;131
8.4.2;2. Different Microarray Platforms;132
8.4.3;3. Experimental Design;136
8.4.4;4. Experimental Variation;137
8.4.5;5. Sample Preparation;140
8.4.6;6. Microarray Preprocessing;148
8.4.7;7. Clustering;154
8.4.8;8. Differential Expression Analysis;159
8.4.9;9. Data Analysis: Understanding the Perturbation;161
8.4.10;10. Closing Remarks;163
8.4.11;References;164
8.5;Chapter 5: Orthogonal Gene Expression in Escherichia coli;170
8.5.1;1. Introduction;171
8.5.2;2. High-Throughput Screening for Orthogonal T7 Promoter O-rbs System;174
8.5.3;3. Integration of Orthogonal Pairs to Synthesize Transcription-Translation FFL;177
8.5.4;4. Engineering the FFL Delay via the Discovery of a Minimal O-rRNA;178
8.5.5;5. Discussion;181
8.5.6;6. Material and Methods;184
8.5.7;Acknowledgments;187
8.5.8;References;187
8.6;Chapter 6: Directed Evolution of Promoters and Tandem Gene Arrays for Customizing RNA Synthesis Rates and Regulation;190
8.6.1;1. Introduction;191
8.6.2;2. Promoter Modification by Error-Prone PCR;193
8.6.3;3. Generating Stable Tandem Gene Arrays for Controlling RNA Synthesis Rate;205
8.6.4;4. Concluding Remarks;208
8.6.5;References;209
9;Section Two: Device and System Design, Optimization, and Debugging;212
9.1;Chapter 7: Design and Connection of Robust Genetic Circuits;214
9.1.1;1. Introduction;215
9.1.2;2. Sources of Failure;216
9.1.3;3. Robustness Principles and Examples in Natural Systems;218
9.1.4;4. Methods for Obtaining Robust Synthetic Circuits;220
9.1.5;5. Robustness Trade-Offs;236
9.1.6;6. Conclusion;237
9.1.7;References;237
9.2;Chapter 8: Engineering RNAi Circuits;242
9.2.1;1. Introduction;243
9.2.2;2. Constructing a Computational Logic Core for the RNAi-Based DNF Circuit;244
9.2.3;3. Constructing a Computational Logic Core for the RNAi-Based CNF Circuit;255
9.2.4;4. Transition from siRNA to miRNA;257
9.2.5;Acknowledgments;259
9.2.6;References;259
9.3;Chapter 9: From SELEX to Cell...;262
9.3.1;1. Introduction;262
9.3.2;2. General Precautions;263
9.3.3;3. In Vitro Selection;263
9.3.4;4. In Vivo Selection;267
9.3.5;References;274
9.4;Chapter 10: Using Noisy Gene Expression Mediated by Engineered Adenovirus to Probe Signaling Dynamics in Mammalian Cells;276
9.4.1;1. Introduction;277
9.4.2;2. Design and Construction;279
9.4.3;3. Measurement;285
9.4.4;4. Broader Applications;289
9.4.5;References;289
9.5;Chapter 11: De novo Design and Construction of an Inducible Gene Expression System in Mammalian Cells;294
9.5.1;1. Introduction;295
9.5.2;2. Selection of a Conditional DNA-Binding Protein;298
9.5.3;3. Establishment of the Inducible Expression System;299
9.5.4;4. Optimization of the Expression System;303
9.5.5;5. Summary;305
9.5.6;Acknowledgments;306
9.5.7;References;306
9.6;Chapter 12: BioBuilding...;310
9.6.1;1. Introduction;311
9.6.2;2. Eau d'coli;312
9.6.3;3. "Eau That Smell" Teaching Lab Using the MIT iGEM Team's Eau d'coli Cells;314
9.6.4;4. Teaching Labs Modified for Resource-Stretched Settings;322
9.6.5;5. Summary;324
9.6.6;Acknowledgments;325
9.6.7;References;325
10;Section Three: Device Measurement, Optimization, and Debugging;328
10.1;Chapter 13: Use of Fluorescence Microscopy to Analyze Genetic Circuit Dynamics;330
10.1.1;1. Fluorescent Reporters;331
10.1.2;2. Constructing and Using Genetic Fluorescent Reporters;332
10.1.3;3. Fluorescent Time-Lapse Microscopy;336
10.1.4;4. Measuring and Interpreting Dynamics;339
10.1.5;5. Applications for Measurement of Circuit Dynamics;341
10.1.6;References;347
10.2;Chapter 14: Microfluidics for Synthetic Biology;350
10.2.1;1. Part I: Introduction;351
10.2.2;2. Part II: Fabrication;393
10.2.3;3. Part III: Experiments;413
10.2.4;Appendix;424
10.2.5;Acknowledgments;426
10.2.6;References;426
10.3;Chapter 15: Plate-Based Assays for Light-Regulated Gene Expression Systems;428
10.3.1;1. Bacterial Photography Protocol;429
10.3.2;2. Bacterial Edge Detection Protocol;434
10.3.3;3. Setting up a Projector-Incubator;436
10.3.4;4. The beta-Galactosidase/S-Gal Reporter System;439
10.3.5;5. Quantifying Signal Intensity on the Plates;440
10.3.6;6. Microscopic Imaging of Agarose Slabs;440
10.3.7;7. Properties of Relevant Strains;441
10.3.8;8. Properties of Relevant Plasmids;442
10.3.9;References;445
10.4;Chapter 16: Spatiotemporal Control of Small GTPases with Light Using the LOV Domain;448
10.4.1;1. Introduction;449
10.4.2;2. The LOV Domain as a Tool for Protein Caging;450
10.4.3;3. Design and Structure Optimization of PA-Rac;450
10.4.4;4. Activation of PA-Rac in Living Cells;452
10.4.5;5. Application of PA-Rac in Drosophila Ovarian Border Cell Migration;455
10.4.6;References;462
10.5;Chapter 17: Light Control of Plasma Membrane Recruitment Using the Phy-PIF System;464
10.5.1;1. Introduction;465
10.5.2;2. Light-Controlled Phy-PIF Interaction;466
10.5.3;3. Genetic Constructs Encoding Phy and PIF Components;467
10.5.4;4. Purification of PCB from Spirulina;470
10.5.5;5. Cell Culture Preparation for Phy-PIF Translocation;473
10.5.6;6. Imaging PIF Translocation Using Spinning Disk Confocal Microscopy;474
10.5.7;Acknowledgments;476
10.5.8;References;476
10.6;Chapter 18: Synthetic Physiology...;480
10.6.1;1. Introduction;481
10.6.2;2. Molecular Design and Construction;484
10.6.3;3. Transduction of Microbial Opsins into Cells for Heterologous Expression;487
10.6.4;4. Physiological Assays;490
10.6.5;5. Conclusion;493
10.6.6;Acknowledgments;494
10.6.7;References;494
11;Section Four: Devices for Metabolic Engineering;500
11.1;Chapter 19: Metabolic Pathway Flux Enhancement by Synthetic Protein Scaffolding;502
11.1.1;1. Introduction;503
11.1.2;2. Method-How to Build Modular Protein Scaffolded Systems for Metabolic Engineering Applications;509
11.1.3;3. Systems that May Benefit from Scaffolding;520
11.1.4;4. Concluding Remarks;520
11.1.5;Acknowledgments;521
11.1.6;References;521
11.2;Chapter 20: A Synthetic Iterative Pathway for Ketoacid Elongation;524
11.2.1;1. Introduction;525
11.2.2;2. Natural Pathways Involving Ketoacid Chain Elongations Catalyzed by the LeuABCD-Dependent Mechanisms;526
11.2.3;3. IPMS and Similar Enzymes;528
11.2.4;4. Expansion to Nonnatural Pathways;530
11.2.5;5. Transfer of Citramalate Pathway to E. coli for Ketoacid Chain Elongation;533
11.2.6;6. Conclusion Remarks;535
11.2.7;References;535
12;Section Five: Expanding Chassis;538
12.1;Chapter 21: Synthetic Biology in Streptomyces Bacteria;540
12.1.1;1. Synthetic Biology for Novel Compound Discovery in Streptomyces;541
12.1.2;2. Practical Considerations for Synthetic Biology in Streptomyces;543
12.1.3;3. Iterative Reengineering of Secondary Metabolite Gene Clusters;544
12.1.4;4. The Molecular Toolbox for Streptomyces Synthetic Biology;546
12.1.5;5. Transcriptional Control;547
12.1.6;6. Translational Control;549
12.1.7;7. Vectors;549
12.1.8;Acknowledgments;552
12.1.9;References;552
12.2;Chapter 22: Methods for Engineering Sulfate Reducing Bacteria of the Genus Desulfovibrio;558
12.2.1;1. Introduction;559
12.2.2;2. Chromosomal Modifications Through Homologous Recombination;560
12.2.3;3. Culturing Conditions and Antibiotic Selection;562
12.2.4;4. DNA Transformation;565
12.2.5;5. Screening Colonies for Proper Integration;568
12.2.6;6. Complementing Gene Deletions;569
12.2.7;7 Concluding Remarks;570
12.2.8;Acknowledgments;571
12.2.9;References;571
12.3;Chapter 23: Modification of the Genome of Rhodobacter sphaeroides and Construction of Synthetic Operons;574
12.3.1;1. Introduction;575
12.3.2;2. Gene Disruption and Deletion;577
12.3.3;3. Construction of Synthetic Operons;582
12.3.4;4. Future Directions;587
12.3.5;References;588
12.4;Chapter 24: Synthetic Biology in Cyanobacteria...;594
12.4.1;1. Introduction;595
12.4.2;2. Cyanobacterial Chassis;597
12.4.3;3. Biological Parts in Cyanobacteria;599
12.4.4;4. Genetic Engineering of Cyanobacteria;605
12.4.5;5. Molecular Analysis of Cyanobacteria;617
12.4.6;6. Conclusion and Outlook;626
12.4.7;Acknowledgments;627
12.4.8;References;627
12.5;Chapter 25: Developing a Synthetic Signal Transduction System in Plants;636
12.5.1;1. Introduction;637
12.5.2;2. Foundation for Developing a Molecular Testing Platform for HK Systems;641
12.5.3;3. Technical Considerations in Developing a Eukaryotic Synthetic Signal Transduction System Based on Bacterial TCS Components.;644
12.5.4;4. A Partial Synthetic Signal Transduction System Using Cytokinin Input;647
12.5.5;5. A Eukaryotic Synthetic Signal Transduction Pathway;648
12.5.6;6. Conclusions;650
12.5.7;7. Protocols;652
12.5.8;Acknowledgments;654
12.5.9;References;654
12.6;Chapter 26: Lentiviral Vectors to Study Stochastic Noise in Gene Expression;658
12.6.1;1. Introduction;659
12.6.2;2. The Lentiviral-Vector Approach;660
12.6.3;3. Production of Lentiviral Vectors and Transduced Cell Lines;664
12.6.4;4. Procedure for Constructing a CV2 Versus Mean Plot;671
12.6.5;5. Inferring Promoter Regulatory Architecture from CV2 Versus Mean Analysis;671
12.6.6;6. Conclusion;675
12.6.7;Acknowledgments;675
12.6.8;References;675
13;Author Index;678
14;Subject Index;706
15;Colour Plate;718