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Riboswitches as Targets and Tools
1st Edition - January 12, 2015
Editor: Donald H. Burke-Aguero
Hardback ISBN:9780128011232
9 7 8 - 0 - 1 2 - 8 0 1 1 2 3 - 2
eBook ISBN:9780128013366
9 7 8 - 0 - 1 2 - 8 0 1 3 3 6 - 6
This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in… Read more
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This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in riboswitches as targets and tools and contains sections on such topics as constructing and optimizing artificial riboswitches, live cell imaging and intracellular sensors with artificial riboswitches, conditional control of gene expression with artificial riboswitches, using artificial riboswitches for protein evolution and pathway optimization, and anti-riboswitch drug screens.
Continues the legacy of this premier serial with quality chapters authored by leaders in the field
Covers research methods in riboswitches as targets and tools
Contains sections on such topics as constructing and optimizing artificial riboswitches, synthetic biology: live cell imaging and intracellular sensors with artificial riboswitches, synthetic biology: conditional control of gene expression with artificial riboswitches, synthetic biology: using artificial riboswitches for protein evolution and pathway optimization, anti-riboswitches drug screens
Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists.
Preface
Volume 1
Volume 2
Chapter One: Design of Transcription Regulating Riboswitches
Abstract
1 Introduction
2 Computational Design of RNA Structures
3 Experimental Evaluation of Designed RNA Structures
4 Concluding Remarks
Chapter Two: Ligand-Dependent Exponential Amplification of Self-Replicating RNA Enzymes
Abstract
1 Introduction
2 Exponential Amplification of RNA Enzymes
3 Ligand-Dependent Exponential Amplification
4 Nuclease-Resistant Autocatalytic Aptazymes
5 Real-Time Fluorescence Assays
6 Conclusions
Acknowledgments
Chapter Three: Design of Modular “Plug-and-Play” Expression Platforms Derived from Natural Riboswitches for Engineering Novel Genetically Encodable RNA Regulatory Devices
Abstract
1 Introduction
2 Design of Riboswitch Modules
3 Analysis of Riboswitch Activity Using an In Vitro Single-Turnover Transcription Assay
4 Cell-Based GFP Reporter Assay
5 Concluding Remarks
Acknowledgment
Chapter Four: Integrating and Amplifying Signal from Riboswitch Biosensors
Abstract
1 Introduction
2 Riboswitch Signal Integration
3 Riboswitch Signal Amplification Using Biological Circuitry
Acknowledgments
Chapter Five: Simple Identification of Two Causes of Noise in an Aptazyme System by Monitoring Cell-Free Transcription
Abstract
1 Theory
2 Equipment
3 Materials
4 Solutions and Buffers
5 Protocol
6 Step 1: Cell-Free Transcription–Translation and Fluorescence Monitoring
7 Step 2: Data Analysis
8 Step 3: (Optional) Quantification of the Intermediate RNAs
Chapter Six: Engineering of Ribosomal Shunt-Modulating Eukaryotic ON Riboswitches by Using a Cell-Free Translation System
Abstract
1 Introduction
2 A Eukaryotic Translation Mechanism Requiring a Rigid mRNA Structure for Ribosomal Progression
3 Choice of a Translation System for Engineering Artificial Riboswitches
4 In Vitro-Selected Aptamer for Ribosomal Shunt-Modulating Riboswitches
5 How to Implant the Selected Aptamer into mRNA
6 General Design of mRNAs with Ribosomal Shunt-Modulating Riboswitches
7 Experiments
8 Conclusion
Acknowledgments
Chapter Seven: Live-Cell Imaging of Mammalian RNAs with Spinach2
Abstract
1 Introduction
2 Developing Spinach, an RNA Mimic of GFP
3 Imaging with Spinach2, a Superfolding Variant of Spinach
4 Fluorescence Imaging of Spinach2-Tagged RNAs
5 Imaging Other RNAs Using Spinach2
Acknowledgments
Chapter Eight: In Vitro Analysis of Riboswitch–Spinach Aptamer Fusions as Metabolite-Sensing Fluorescent Biosensors
Abstract
1 Introduction
2 Design and Preparation of an RNA-Based Fluorescent Biosensor
3 Determination of Ligand Selectivity and Affinity of Biosensor by Fluorescence Activation
4 Determination of Binding Kinetics of Biosensor
Chapter Nine: Using Spinach Aptamer to Correlate mRNA and Protein Levels in Escherichia coli
Abstract
1 Introduction
2 Parts Selection and Plasmid Construction
3 E. coli Strain Selection
4 Culturing and Inducing E. coli Cells
5 Correlating mRNA and Protein Production Using Flow Cytometry
6 Correlating mRNA and Protein Production Using Fluorescence Microscopy
7 Summary
Acknowledgments
Chapter Ten: Monitoring mRNA and Protein Levels in Bulk and in Model Vesicle-Based Artificial Cells
Abstract
1 Introduction
2 The “Spinach Technology” for Combined Detection of mRNA and Protein in Cell-Free Expression Systems
3 Quantifying the Levels of mRNA and Protein Synthesized in PURE System Bulk Reactions