Biological Engineering with Chemical-Sensing MacromolecularSwitches: I. Discovery and Applications of Small-Molecule DependentSynthetic Riboswitches II. A Genetic Toolbox for CreatingReversible Ca 2+ -Sensitive Biomaterials Open Access

Abstract

Nature has evolved the ability to precisely coordinate physiological and cellular processes in response to a variety of chemical signals.This dissertation draws inspiration from the exquisite chemical-sensing abilities of natural macromolecules toward reengineering chemical-sensing systems for applications in synthetic biology or nanotechnology.

Part I focuses on the development of efficient methods to select for synthetic riboswitches, and the use of these genetic control elements to modulate complex bacterial behavior. In Chapter 2, we demonstrate that synthetic riboswitches can be used to regulate E. coli chemotaxis with an exogenous ligand that wild-type cells neither recognize as a chemoattractant, nor naturally detect. The reprogrammed cells can be guided toward and precisely localized to a completely new chemical signal. Chapter 3 presents the development of a high-throughput selection to identify synthetic riboswitches by selecting for cells that exhibit ligand-dependent changes in migration on semi-solid media. We also discuss complications of this method and present potential solutions to surmount these limitations. Chapter 4 discusses studies toward overcoming our previously unproductive efforts to identify synthetic riboswitches that could repress bacterial gene expression when a small-molecule ligand is provided. These studies revealed a novel mechanism by which synthetic riboswitches may function in E. coli cells. In Chapter 5, we present principles to introduce synthetic riboswitches into a diverse set of prokaryotes. For species lacking dynamic inducible promoter systems, the introduction of synthetic riboswitch technologies will facilitate previously intractable genetic and biochemical studies.

Part II focuses on our efforts to develop ‘smart' materials that sense specific chemical signals in complex environments and respond with predictable changes in their mechanical properties. Toward this end, we developed a genetic toolbox of natural and engineered protein modules that can be rationally combined in many ways to create reversible self-assembling materials that vary in their composition, architecture, and mechanical properties. Using this toolbox, we produced and characterized several materials that reversibly self-assemble in the presence of calcium ions. The properties of these materials could be predicted from the dilute solution behavior of their component modules, suggesting that this toolbox may be generally useful for creating new stimuli-sensitive materials.

Table of Contents

Part I: Discovery and Applications of Small-Molecule Dependent Synthetic Riboswitches...1 Chapter 1: Introduction...2 1.1 Chemical Sensing is a Fundamental Property of Life...2 1.2 Metabolite-Sensing can be Achieved Using RNA...4 1.3 Synthetic Riboswitches...6 1.4 References...10 Chapter 2: Guiding E. coli with Small Molecules and RNA...12 2.1 Introduction...12 2.2 Results and Discussion...16

2.2.1 Regulating CheZ Expression with a Theophylline-Dependent Riboswitch...16 2.2.2 Ligand-Dependent Migration of E. coli Populations on Semi-Solid Agar...17 2.2.3 Testing the Gradient-Sensing Abilities of Reprogrammed Cells...18 2.2.4 Quantifying the Behavior of Reprogrammed Cells using Light Microscopy...20 2.2.5 Patterning of Reprogrammed Cells on Semi-Solid Media...22 2.2.6 Changing the Ligand Specificity to 3-Methylxanthine...24

2.3 Conclusion...25 2.4 Experimental...26 2.5 References...33 Chapter 3: Riboswitches on the Move: A High-Throughput Selection Based on Cell Motility...35 3.1 Introduction...35 3.2 Results and Discussion...37

3.2.1 Pre-selection for RBS Sequences that Provide Optimal CheZ Levels...37 3.2.2 A Library for Motility Selections...41 3.2.3 Motility Selections with Two Different Promoters...42 3.2.4 Confirmation of Selected Riboswitches...45 3.2.5 Monitoring the Progression of a Library under Selective Pressure...47 3.2.6 Ligand-Dependent Motility of Cells Harboring Selected Riboswitches...48 3.2.7 A Correlation Between Migration Distance and β-Galactosidase Activity...49

3.3 Conclusion...51 3.4 Experimental...51 3.5 References...56 Chapter 4: Switching the Switch: An Unexpected Mechanism for Small-Molecule Dependent Translational Repression...57 4.1 Introduction...57 4.2 Results and Discussion...58

4.2.1 Designing a Combinatorial Library for Ligand-Dependent Repression...59 4.2.2 A Redesigned Library Positions the RBS within the Aptamer Stem...63 4.2.3 Removal of the ATG codon 3′ to the Aptamer Improves Gene Repression...65 4.2.4 The Presence and Position of the 5′ ATG Is Critical for Riboswitch Function...69 4.2.5 Mass Spectrometry Shows that the Riboswitch Is Translated...73 4.2.6 RNA Structure Probing Studies of the 27-Fold Repressor...76 4.2.7 Repressor Switches Complement Previously Identified Riboswitches...84

4.3 Conclusion...86 4.4 Experimental...86 4.5 References...96 Chapter 5: Exploring the Portability of Synthetic Riboswitches for use as Genetic Tools in Diverse Bacterial Species...98 5.1 Introduction...98 5.2 Results and Discussion...101

5.2.1 Addressing the Conundrum of Riboswitches in Acinetobacter baylyi...101 5.2.2 Selection of the Study Species...103 5.2.3 Testing Previously Identified Riboswitches in Several Species...105 5.2.4 Semi-Rational Riboswitches for Gram-Positive Bacteria...107 5.2.5 Testing a ‘Riboswitch Package' in Mycobacterium smegmatis...110

5.3 Conclusion...119 5.4 Experimental...120 5.5 References...125

Part II: A Genetic Toolbox for Creating Reversible Ca²⁺-Sensitive Biomaterials...128 Chapter 6: A Genetic Toolbox for Creating Reversible Ca^2+-Sensitive Biomaterials...129 6.1 Introduction...129 6.2 Results and Discussion...133

6.2.1 A Genetic Toolbox for Materials Design...133 6.2.2 Varying the Stoichiometry of Junction Points...137 6.2.3 Varying the Binding Strength of Junction Points...144

6.3 Conclusion...148 6.4 Experimental...149 6.5 References...156

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Biology, Molecular Engineering, Chemical
Keyword	RNA riboswitch high-throughput screening biomaterials chemotaxis synthetic biology
Committee Chair / Thesis Advisor	Gallivan, Justin, Emory University
Committee Members	Conticello, Vincent, Emory University Lynn, David, Emory University

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