UID:
almahu_9949697833402882
Umfang:
1 online resource (468 p.)
ISBN:
0-12-801336-2
,
0-12-801123-8
Serie:
Methods in Enzymology ; Volume 550
Inhalt:
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 se
Anmerkung:
Description based upon print version of record.
,
Front Cover; Riboswitches as Targets and Tools; Copyright; Contents; Contributors; Preface; Volume 1; Volume 2; Chapter 1: Design of Transcription Regulating Riboswitches; 1. Introduction; 2. Computational Design of RNA Structures; 2.1. The inverse folding problem; 2.2. Designing multi-stable RNAs; 2.3. Modeling external triggers; 2.4. Current limitation of design software; 3. Experimental Evaluation of Designed RNA Structures; 3.1. Considerations on candidate selection and cloning procedures; 3.2. Further characterization and current limitations; 4. Concluding Remarks; References
,
Chapter 2: Ligand-Dependent Exponential Amplification of Self-Replicating RNA Enzymes1. Introduction; 2. Exponential Amplification of RNA Enzymes; 2.1. Materials; 2.2. Procedure for RNA self-replication; 3. Ligand-Dependent Exponential Amplification; 3.1. Procedure for quantitative ligand detection; 3.2. Multiplexed ligand detection; 3.3. Coupling ligand recognition to ligand-independent amplification; 3.4. Procedure for coupled amplification; 4. Nuclease-Resistant Autocatalytic Aptazymes; 5. Real-Time Fluorescence Assays; 6. Conclusions; Acknowledgments; References
,
Chapter 3: Design of Modular ``Plug-and-Play ́ ́ Expression Platforms Derived from Natural Riboswitches for Engineering Nov...1. Introduction; 2. Design of Riboswitch Modules; 2.1. Design strategy; 2.2. Design optimization; 3. Analysis of Riboswitch Activity Using an In Vitro Single-Turnover Transcription Assay; 3.1. Template construction; 3.1.1. Overlapping extension PCR; 3.1.2. Purification of the template; 3.2. Single-turnover in vitro transcription assay; 4. Cell-Based GFP Reporter Assay; 4.1. Reporter design; 4.2. Protocol; 4.3. Other considerations using in vivo reporters
,
5. Concluding RemarksAcknowledgment; References; Chapter 4: Integrating and Amplifying Signal from Riboswitch Biosensors; 1. Introduction; 1.1. Biological circuits; 2. Riboswitch Signal Integration; 2.1. Design; 2.1.1. AND gate; 2.1.2. Selection of riboswitches; 2.1.3. Using plasmid backbones available from the Registry of Standard Biological Parts; 2.2. Build; 2.2.1. Materials for construction of a riboswitch-based AND logic gate; 2.2.2. Methods to construct pANDrs; 2.3. Test; 3. Riboswitch Signal Amplification Using Biological Circuitry; 3.1. Design; 3.2. Build
,
3.2.1. Materials for the construction of an amplification circuit3.2.2. Methods to construct an amplification circuit, pAMPv1; 3.2.2.1. PLas_RBS34(strong)_GFPa1_Terminator_Weak RBS_LasI +/-degradation tag; 3.3. Test; 3.4. Redesign and build; 3.4.1. Methods to construct amplification circuit version 2; 3.4.1.1. pAMPv2.1: PLas_RBS 34 (strong)_GFPa1_Terminator_RBS 33 (very weak)_RhlILVA; 3.4.1.2. pAMPv2.2: PRhl_RBS 34 (strong)_GFPa1_Terminator_RBS 33 (very weak)_LasILVA; 3.5. Test; 3.5.1. Fluorescence activation; 3.5.2. Signal progression; Acknowledgments; References
,
Chapter 5: Simple Identification of Two Causes of Noise in an Aptazyme System by Monitoring Cell-Free Transcription
,
English
Weitere Ausg.:
ISBN 1-336-00896-2
Sprache:
Englisch
