Biological engineering with chemical-sensing macromolecular switches: I. Discovery and applications of small-molecule dependent synthetic riboswitches II. A genetic toolbox for creating reversible calcium-sensitive biomaterials.

摘要

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.