| 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. |
