The Design of Theophylline Sensing Riboswitches in Agrobacteriumtumefaciens and Their Application in Reprogramming theVirA/VirG Two-Component System Open Access

Abstract

Abstract The Design of Theophylline Sensing Riboswitches in Agrobacterium tumefaciens and Their Application in Reprogramming the VirA/VirG Two-Component System By Ian Goldlust

Bacteria have evolved several mechanisms to sense and respond to chemical stimuli. One such mechanism, the riboswitch, is a cis-acting regulatory RNA that can bind metabolites and subsequently control gene expression and modify an organism's behavior.

Chapter one starts with an overview of synthetic and natural riboswitches and their mechanism of action. Chapter two presents a strategy to design and optimize synthetic theophylline-sensing riboswitches for the α-proteobacteria Agrobacterium tumefaciens - starting from weakly functioning Escherichia coli-optimized riboswitches, and moving towards the discovery a 19-fold riboswitch. Chapter three discusses the potential for increasing riboswitch function through the incorporation of an epsilon (ε) enhancer sequence into the expression platform. Finally, Chapter four begins by outlining a strate-gy to reprogram the VirA/VirG, two-component system of A. tumefaciens so that it re-sponds only to the small molecule theophylline and ends by discussing a strategy to transport this system effectively into E. coli.

Table of Contents

Contents

Chapter 1: Introduction...1

Chapter 2: The Design and Optimization of Transportable Synthetic Riboswitches in Agrobacterium Tumefaciens...6

2.1 Introduction...6

2.2 Results and Discussion...7

2.2.1 Testing Previously Identified E. coli Riboswitches in A. tumefaciens...7

2.2.2 Testing Previously Identified A. baylyi Riboswitches in A. tumefaciens...8

2.2.3 Low-Throughput Screening and Testing of Previously Identified Gram-Positive Riboswitches in A. tumefaciens...9

2.2.4 Towards Identifying Better Switches in A. tumefaciens...10

2.3 Conclusion...13

2.4 Experimental...15

Chapter 3: Using Epsilon (ε) Sequences to Enhance the Activation of Synthetic Riboswitches...20

3.1 Introduction...20

3.2 Results and Discussion...22

3.3 Conclusion...23

3.4 Experimental...25

Chapter 4: Hijacking and Reprogramming the VirA/VirG Two-Component Sys-tem of Agrobacterium tumefaciens...26

4.1 Abstract...26 4.2 Introduction...27

4.2.1 The Virulence System of A. tumefaciens...27

4.3 Experimental Design...29

4.3.1 Controlling the Virulence Pathway Using the Single Clone Riboswitch...29

4.3.2 Additional Library Screens to Isolate Optimized Riboswitch for Controlling Virulence...30

List of Primers Used...31

References...33

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	MS
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Biology, Microbiology
Keyword	Two-component system Riboswitch Theophylline Agrobacteria
Committee Chair / Thesis Advisor	Gallivan, Justin, Emory University
Committee Members	Lutz, Stefan, Emory University Lynn, David, Emory University

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