Engineering Control of Gene Expression in Bacteria Using RNA-Small Molecule Interactions Público

Desai, Shawn K (2008)

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Small molecule-responsive riboswitches, which are composed entirely of RNA and control gene expression, have recently been discovered in a variety of organisms, including bacteria. These regulators have all the attributes of protein-based sensors, but are less complex and smaller than their protein counterparts. Because of their relatively simple design and broad recognition capabilities, many researchers are interested in constructing riboswitches in bacteria that respond to small molecules of their choosing. Once assembled, these engineered bacteria could be used for a variety of applications, such as sensing landmines, or for directing cancer-killing bacteria to cancerous cells.

In this thesis, we present our research directed, broadly, toward engineering small molecule-responsive riboswitches in bacteria. Chapter 2 describes our successful construction of a small molecule-responsive synthetic riboswitch in Escherichia coli, followed by a thorough characterization of its mechanism. After determining that it operates by activating translation, we used the riboswitch in both screens and selections for small molecules.

Chapter 3 presents the development and application of high-throughput screens and selections for synthetic riboswitches. We used these techniques to discover synthetic riboswitches in bacteria with outstanding activation ratios, and sequencing of the resulting variants allowed us to propose a potential model for their function. Several studies were undertaken to test the model, and it appears to be correct.

In Chapter 4, we explore the transferability of synthetic riboswitches between the similarly related organisms, E. coli and Acinetobacter baylyi. We chose A. baylyi because it has a number of traits that E. coli does not have, such as natural competence, and we wished to enable the conditional control of gene expression in this potentially useful organism. We also adapted our previous high-throughput screen for use with A. baylyi, and used it to identify several synthetic riboswitches with large activation ratios.

Chapter 5 describes our efforts toward using a synthetic riboswitch-mediated auxotroph to functionally clone an unknown enzyme from Coffea arabica that catalyzes the rate-limiting step in caffeine catabolism.

Finally, Chapter 6 presents our attempts at constructing synthetic riboswitches that respond to small molecules involved in plant isoquinoline alkaloid biosynthesis.

Table of Contents

Chapter 1 Introduction: Natural and Synthetic Control of Gene Expression Using Small Molecules 1 1.1 Small Molecules Can Regulate Gene Expression 2 1.1.1 The LacI Protein 4 1.2 Riboswitches 5 1.2.1 Discovery 5 1.2.2 Genetic Control Mechanisms 7 1.2.3 Structural Characteristics 12 1.3 Engineering Small Molecule Control of Gene Expression 14 1.3.1 Engineering Protein-Small Molecule Interactions to Control Gene Expression 14 1.3.2 Engineering RNA-Small Molecule Interactions to Control Gene Expression 22 1.4 Conclusion 29 1.5 References 30 Chapter 2 Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch that Activates Protein Translation 37 2.1 Introduction 38 2.2 Results and Discussion 40 2.2.1 Creation of a Synthetic Riboswitch 40 2.2.2 Determining the Mechanism of Action 45 2.2.3 Genetic Selections for Small Molecules Using a Synthetic Riboswitch 52 2.2.4 Genetic Screens and Selections to Discover Synthetic Riboswitches in E. coli 54 2.3 Conclusion 56 2.4 Experimental 57 2.5 References 66 Chapter 3 High-Throughput Screens and Selections for Synthetic Riboswitches 69 3.1 Introduction 70 3.2 Results and Discussion 72 3.2.1 Creation of a Library of Randomized Mutants 72 3.2.2 Selection System for Optimally Functioning Riboswitches 73 3.2.3 High-Throughput "Stamping" Assay for Optimally Functioning Riboswitches 77 3.2.4 Plate-Based Assay for Optimally Functioning Riboswitches 82 3.2.5 Possible Mechanism of Action for Synthetic Riboswitch Function 85 3.2.6 A Model for Synthetic Riboswitch Function 88 3.2.7 Potential Design Implications for Synthetic Riboswitches 94 3.3 Conclusion 95 3.4 Experimental 96 3.5 References 105 Chapter 4 Engineering Ligand-Activated Genetic Control Elements in Acinetobacter baylyi ADP1 107 4.1 Introduction 108 4.2 Results and Discussion 111 4.2.1 Introduction of Synthetic Riboswitches from E. coli into A. baylyi 111 4.2.2 Screening for Synthetic Riboswitches in A. baylyi 116 4.3 Conclusion 120 4.4 Experimental 121 4.5 References 126 Chapter 5 Studies Toward Functional Cloning of a Putative Caffeine N-7 Demethylase from Coffea arabica 128 5.1 Introduction 129 5.2 Results and Discussion 135 5.2.1 Creation of a Theophylline-Selectable cDNA Cloning Vector and a C. arabica cDNA Library 135 5.2.2 Selections for a Putative Caffeine 7-NDM from C. arabica 138 5.2.3 Screening for a Putative Caffeine 7-NDM from C. arabica 144 5.3 Conclusion 145 5.4 Experimental 146 5.5 References 154 Chapter 6 Studies Toward Engineering Synthetic Riboswitches that Respond to (S)-Coclaurine and 2-Methyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline 156 6.1 Introduction 157 6.2 Results and Discussion 160 6.2.1 Optimization of SELEX Procedure 161 6.2.2 Selections for (S)-Coclaurine Aptamers 162 6.2.3 Selections for N-methyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline Aptamers 165 6.2.4 Biochemical Analysis of Putative (S)-Coclaurine Binding RNA Aptamers 166 6.2.5 Converting a Single Putative (S)-Coclaurine Binding Aptamer into a Synthetic Riboswitch 168 6.2.6 Identifying a Synthetic Riboswitch from a Pool of Potential Aptamers 170 6.2.7 Identifying an (S)-Coclaurine Riboswitch Using a Novel Strategy 170 6.3 Conclusion 174 6.4 Experimental 175 6.5 References 185

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