Synthetic Biology: Methods for Part Device Characterization and Chassis Engineering

Series editors......Page 1
Copyright......Page 2
Contributors
......Page 3
Preface......Page 11
Sequence-Specificity and Energy Landscapes of DNA-Binding Molecules......Page 13
Introduction......Page 14
Labeling DNA-binding molecules......Page 16
Synthetic DNA-binding molecules......Page 19
Natural or engineered DNA-binding proteins......Page 20
Label-free detection......Page 22
Solution-Based Cognate Sequence Identification......Page 23
Sequence-specificity landscapes......Page 28
Binding energy landscapes......Page 33
Genomescapes......Page 35
References......Page 37
Promoter Reliability in Modular Transcriptional Networks......Page 41
Measuring the dynamics of an embedded network......Page 43
Modeling an embedded positive-feedback module......Page 46
Promoter properties depend on copy number......Page 49
Operator buffers insulate promoter properties from context......Page 51
Conclusion......Page 54
Strains and media......Page 55
Dynamic measurements......Page 56
References......Page 57
The Analysis of ChIP-Seq Data......Page 60
Introduction......Page 61
Choices of sequencing platforms......Page 62
Sequencing statistics and quality control......Page 64
Saturation......Page 65
Negative controls......Page 66
Processing and Analyzing ChIP-Seq Datasets......Page 67
Step 1. Map the reads back to the reference genome......Page 68
Step 2. Background estimation......Page 69
Step 3. Peak calling......Page 71
Step 4. Gene assignment and peak annotation......Page 75
Step 5. De novo motif analysis......Page 76
Discussion......Page 77
Acknowledgments......Page 79
References......Page 80
Using DNA Microarrays to Assay Part Function......Page 83
Introduction......Page 84
Different Microarray Platforms......Page 85
Experimental Design......Page 89
Experimental Variation......Page 90
Solutions......Page 93
Sample harvesting......Page 94
Total RNA preparation......Page 95
Assessing RNA quality and yield......Page 96
Sample cleanup......Page 97
Cy3/Cy5 coupling......Page 98
Sample hybridization......Page 99
Slide washing and scanning......Page 100
Microarray Preprocessing......Page 101
Diagnostic plots of gene expression ratios......Page 102
Data normalization......Page 105
Clustering......Page 107
Measures of similarity between genes and distance between clusters......Page 108
Clustering algorithms......Page 109
Differential Expression Analysis......Page 112
Data Analysis: Understanding the Perturbation......Page 114
Closing Remarks......Page 116
References......Page 117
Orthogonal Gene Expression in Escherichia coli......Page 122
Discovery of orthogonal ribosome-orthogonal mRNA pairs......Page 123
Integration of orthogonal transcription-translation pairs into gene expression networks......Page 125
High-Throughput Screening for Orthogonal T7 Promoter O-rbs System......Page 126
Integration of Orthogonal Pairs to Synthesize Transcription-Translation FFL......Page 129
Engineering the FFL Delay via the Discovery of a Minimal O-rRNA......Page 130
Discussion......Page 133
Selection of an optimized T7 promoter/O-rbs system......Page 136
Characterizing pT7 O-rbs GFP expression constructs......Page 137
Characterization of orthogonal gene expression kinetics......Page 138
References......Page 139
Directed Evolution of Promoters and Tandem Gene Arrays for Customizing RNA Synthesis Rates and Regulation......Page 142
Introduction......Page 143
Promoter Modification by Error-Prone PCR......Page 145
Constitutive promoter considerations......Page 146
Inducible promoter considerations......Page 147
Error-prone PCR of promoter......Page 148
S. cerevisiae promoter library cloning......Page 149
Initial selection and quality control......Page 150
E. coli constitutive promoter characterization......Page 151
Growth-phase yECitrine fluorescence......Page 152
S. cerevisiae constitutive promoter characterization......Page 153
Growth-phase yECitrine fluorescence......Page 154
Evolving promoter to increase oxygen EC50......Page 155
Generating Stable Tandem Gene Arrays for Controlling RNA Synthesis Rate......Page 157
Concluding Remarks......Page 160
References......Page 161
Design and Connection of Robust Genetic Circuits......Page 163
Introduction......Page 164
Sources of Failure......Page 165
Robustness Principles and Examples in Natural Systems......Page 167
Improved genetic stability......Page 169
Basic network motifs......Page 171
Modularity......Page 174
Decoupling/orthogonality......Page 176
Redundant pathways/alternative (or fail-safe) mechanisms......Page 178
Intercellular communication......Page 179
Redundancy......Page 180
Noise reduction in multicellular systems......Page 181
Exploiting noise in multicellular systems......Page 182
Circuit construction strategies to improve robustness......Page 183
Robustness Trade-Offs......Page 185
References......Page 186
Engineering RNAi Circuits......Page 191
Introduction......Page 192
Step 1: Design candidate sRNA sequences......Page 193
Step 2: Design a set of siRNAs......Page 195
Step 3: Cloning and experimental characterization of ``computational´´ molecules and the siRNA......Page 196
Cloning protocol for individual siRNA targets (CP1)......Page 197
Testing protocol for individual siRNAs (TP1)......Page 198
Cloning protocol for multitarget constructs (CP2)......Page 201
Step 4: Characterization of the logic core......Page 203
Constructing a Computational Logic Core for the RNAi-Based CNF Circuit......Page 204
Testing protocol for siRNA set for a CNF circuit (TP4)......Page 205
Transition from siRNA to miRNA......Page 206
Testing protocol for miRNA circuits (TP6)......Page 207
References......Page 208
Introduction......Page 210
Materials......Page 211
RNA transcription and purification......Page 213
In vitro selection......Page 214
Material for motility-based selection......Page 215
Generation of library for in vivo selection......Page 216
Motility selection......Page 218
General procedure for motility......Page 219
Procedure for motility selection......Page 220
Enzymatic (beta-galactosidase) screen for functional riboswitches......Page 221
References......Page 222
Using Noisy Gene Expression Mediated by Engineered Adenovirus to Probe Signaling Dynamics in Mammalian Cells......Page 224
Introduction......Page 225
Inputs......Page 227
Adenoviral construction......Page 231
Outputs......Page 232
Infection......Page 233
Analysis......Page 234
Immunolabeling......Page 235
Analysis......Page 236
References......Page 237
De novo Design and Construction of an Inducible Gene Expression System in Mammalian Cells......Page 241
Introduction......Page 242
Selection of a Conditional DNA-Binding Protein......Page 245
Construction of the biotin-responsive promoter......Page 246
Initial testing of the system in cell culture......Page 248
Optimization of the Expression System......Page 250
Integration of the inducible expression system into the desired network......Page 251
Summary......Page 252
References......Page 253
Bio Building: Using Banana-Scented Bacteria to Teach Synthetic Biology......Page 256
Introduction......Page 257
Eau d'coli......Page 258
``Eau That Smell´´ Teaching Lab Using the MIT iGEM Team's Eau d'coli Cells......Page 260
Growing starter cultures for the students......Page 263
Bacterial growth and scent curves......Page 264
Measuring banana-smell and cell growth......Page 265
Assessment......Page 267
Teaching Labs Modified for Resource-Stretched Settings......Page 268
Comparison of growing cultures to Turbidity Standards......Page 269
Summary......Page 270
References......Page 271
Use of Fluorescence Microscopy to Analyze Genetic Circuit Dynamics......Page 273
Fluorescent proteins......Page 274
Transcriptional reporters......Page 275
Translational reporters......Page 277
Introducing genetic reporters into cells......Page 278
Minimal requirements for fluorescence microscopy......Page 279
Tracking lineages......Page 281
Protein concentration and localization......Page 282
Binding interactions FRET, etc.......Page 283
Simplifying interactions comprising cellular differentiation circuits......Page 284
Uncovering novel interactions in genetic circuits......Page 285
Supplementing traditional biochemical promoter analysis......Page 286
Bifurcation of dynamics through parameter perturbation......Page 287
Relationship among genetic circuit architecture, dynamics and biological function......Page 288
References......Page 290
Microfluidics for Synthetic Biology......Page 292
Part I: Introduction......Page 293
The design of a microfluidic chip......Page 295
Mixing in microchemostat devices......Page 296
Calculating flow rates and pressure drops......Page 298
Designing a microchemostat chip......Page 303
Design of an improved DAW junction......Page 313
Calibration of the DAW junction......Page 315
Design of an improved yeast cell trap......Page 317
A parallel DAW device......Page 319
Cell tracking......Page 323
Hardware......Page 331
Software: iDAW......Page 333
Photolithography......Page 335
Photoresist......Page 337
Equipment and environment......Page 339
Photomasks......Page 340
Photolithography: Protocol......Page 341
Dispensing photoresist......Page 343
Alignment of photomask and UV exposure......Page 344
Measuring feature height......Page 345
Protocol......Page 346
Soft lithography......Page 348
Preparing PDMS......Page 349
Degassing PDMS......Page 350
PDMS processing......Page 351
Removing PDMS layer......Page 352
Cleaning chips......Page 353
Troubleshooting......Page 354
Experimental setup for E. coli......Page 355
Wetting the chip......Page 356
Preparing syringes......Page 358
Loading cells......Page 360
Pre-experiment preparation......Page 361
Media preparation......Page 363
Connecting DAW reservoirs to the device......Page 364
Microscope setup......Page 365
Appendix......Page 366
References......Page 368
Plate-Based Assays for Light-Regulated Gene Expression Systems......Page 370
Generation of light-sensing strains......Page 371
Setting up the plate-based assay......Page 372
Two-color bacterial photography......Page 375
Generation of edge detector strains......Page 376
Setting up the plate-based assay......Page 377
Setting up a Projector-Incubator......Page 378
The beta-Galactosidase/S-Gal Reporter System......Page 381
Microscopic Imaging of Agarose Slabs......Page 382
Properties of Relevant Strains......Page 383
pCph8......Page 384
pJT106 and derivatives......Page 385
Green sensor plasmids......Page 386
References......Page 387
Spatiotemporal Control of Small GTPases with Light Using the LOV Domain......Page 389
Introduction......Page 390
Design and Structure Optimization of PA-Rac......Page 391
Cell handling......Page 393
Irradiation in living cells-Light sources, dosage, and spatial control......Page 394
Detection and quantitation of Rac activation in cells......Page 395
What is border cell migration?......Page 396
Genetics......Page 398
Illumination of border cells-In vitro culture, live imaging, and photomanipulation......Page 399
Detection and quantification of effects of Rac activation on border cells......Page 400
What do we learn from PA-Rac application to border cell migration?......Page 402
References......Page 403
Light Control of Plasma Membrane Recruitment Using the Phy-PIF System......Page 404
Introduction......Page 405
Light-Controlled Phy-PIF Interaction......Page 406
Genetic Constructs Encoding Phy and PIF Components......Page 407
Protocol......Page 410
Protocol......Page 413
Imaging PIF Translocation Using Spinning Disk Confocal Microscopy......Page 414
References......Page 416
Synthetic Physiology: Strategies for Adapting Tools from Nature for Genetically Targeted Control of Fast Biological Processes......Page 419
Introduction......Page 420
Molecular Design and Construction......Page 423
Transduction of Microbial Opsins into Cells for Heterologous Expression......Page 426
Physiological Assays......Page 429
Conclusion......Page 432
References......Page 433
Metabolic Pathway Flux Enhancement by Synthetic Protein Scaffolding......Page 438
Introduction......Page 439
Selecting protein-protein interaction domains and ligands for scaffold construction......Page 445
Assembling scaffolds from domains and tagging enzymes for corecruitment......Page 447
Varying scaffold stoichiometry......Page 449
Scaffold composition effects......Page 453
Concluding Remarks......Page 456
References......Page 457
A Synthetic Iterative Pathway for Ketoacid Elongation......Page 460
Introduction......Page 461
Synthesis of leucine (C5C6) and norvaline (C4C5) in Escherichia coli by LeuABCD......Page 462
Reaction mechanisms of carbon-chain elongation catalyzed by LeuABCD......Page 464
Structural and mechanistic homology among LeuA, CimA, and GlcB......Page 465
Iterative ketoacid chain elongation......Page 466
Alteration of LeuA selectivity for longer substrates by rational mutagenesis......Page 467
Directed evolution of 2-ketoacid pathways using amino acid auxotrophs......Page 469
Evolution of citramalate synthase (CimA) for the elongation of pyruvate to 2-ketobutyrate......Page 470
References......Page 471
Synthetic Biology in Streptomyces Bacteria......Page 473
Synthetic Biology for Novel Compound Discovery in Streptomyces......Page 474
Choice of host organism......Page 476
Iterative Reengineering of Secondary Metabolite Gene Clusters......Page 477
Transcriptional control engineering in Streptomyces......Page 478
The Molecular Toolbox for Streptomyces Synthetic Biology......Page 479
Inducible promoters......Page 480
Constitutive promoters......Page 481
Vectors......Page 482
Low copy number vectors......Page 483
References......Page 485
Methods for Engineering Sulfate Reducing Bacteria of the Genus Desulfovibrio......Page 491
Introduction......Page 492
Chromosomal Modifications Through Homologous Recombination......Page 493
Anaerobiosis......Page 495
Culture maintenance......Page 496
Varying the electron donor/acceptor......Page 497
Method for electroporation......Page 498
Southern blot analysis......Page 501
Requirements for complementing plasmid......Page 502
Concluding Remarks......Page 503
References......Page 504
Modification of the Genome of Rhodobacter sphaeroides and Construction of Synthetic Operons......Page 506
Introduction......Page 507
General scheme......Page 509
Construction of the DeltaRCLH mutant......Page 513
Host strain......Page 514
Expression vector......Page 515
Regulation of synthetic operon expression......Page 516
Genome modification......Page 519
References......Page 520
Synthetic Biology in Cyanobacteria: Engineering and Analyzing Novel Functions......Page 526
Introduction......Page 527
Cyanobacterial Chassis......Page 529
Promoters......Page 531
Terminators......Page 532
Ribosome binding sites......Page 533
Antisense RNA......Page 535
Posttranslational control: Degradation tags/proteases......Page 536
Vectors......Page 537
Replicative vectors......Page 538
Integrative vectors......Page 541
Natural transformation......Page 543
Conjugation......Page 544
Electroporation......Page 546
Selection......Page 547
Molecular Analysis of Cyanobacteria......Page 549
Isolation of heterocysts......Page 550
Preparation of DNA......Page 551
Preparation of RNA......Page 552
Preparation of soluble/total proteins......Page 553
Preparation of membrane proteins......Page 554
2D gel electrophoresis: Isoelectric focusing (IEF) and SDS-PAGE......Page 555
Quantitative shotgun proteomics: iTRAQ......Page 556
Gene expression analysis based on reporter proteins......Page 557
Conclusion and Outlook......Page 558
References......Page 559
Developing a Synthetic Signal Transduction System in Plants......Page 567
Signal transduction and synthetic biology......Page 568
Light sensing......Page 569
Cytokinin signaling based on histidine kinases......Page 570
Early work showing plant HKs function in bacteria......Page 572
Development of a synthetic signaling system based on bacterial TCS......Page 574
Technical Considerations in Developing a Eukaryotic Synthetic Signal Transduction System Based on Bacterial TCS Components.......Page 575
Bacterial RRs: Signal dependent nuclear translocation and adaptation for transcriptional activation......Page 576
Consideration of codon bias when designing synthetic signaling systems......Page 577
A Partial Synthetic Signal Transduction System Using Cytokinin Input......Page 578
A Eukaryotic Synthetic Signal Transduction Pathway......Page 579
Conclusions......Page 581
Fluorometric GUS assay (fluorometer)......Page 583
References......Page 585
Lentiviral Vectors to Study Stochastic Noise in Gene Expression......Page 589
Introduction......Page 590
The Lentiviral-Vector Approach......Page 591
Production of Lentiviral Vectors and Transduced Cell Lines......Page 595
Sorting isoclonal populations......Page 599
Inferring Promoter Regulatory Architecture from CV2 Versus Mean Analysis......Page 602
Constitutive promoter architecture......Page 603
Two-state promoter architecture......Page 604
References......Page 606
A......Page 609
B......Page 610
C......Page 612
D......Page 614
F......Page 615
G......Page 616
H......Page 618
I......Page 619
K......Page 620
L......Page 622
M......Page 624
O......Page 626
P......Page 627
R......Page 628
S......Page 629
T......Page 632
V......Page 633
W......Page 634
Y......Page 635
Z......Page 636
Subject Index......Page 637