Modular Riboswitch Reporters for Directed Protein Evolution

University Of Colorado At Boulder, Boulder CO

Investigators

Abstract

With this award, the Chemistry of Life Processes program is supporting the research of Professors Robert Batey and Shelley Copley of the University of Colorado, Boulder. Profs. Batey and Copley will develop a modular and versatile method for creating small molecule biosensors that can be utilized to monitor directed protein evolution. The hypothesis is that one can reverse engineer a form of RNA-based regulation namely "riboswitches" that are found in the leader sequences of mRNAs that directly bind small molecules to direct expression of the message. This proposal aims to exploit these RNAs by two means. First, a "mix-and-match" methodology will be developed that allows a broad array of small molecule receptors to be spliced into biologically efficient regulatory switches that drive in vivo expression of fluorescent protein reporters. Second, in vitro selection methods will be developed to take advantage of the properties of riboswitches to access a new generation of RNA-based small molecule receptors. Finally, these approaches are to be applied to a directed evolution project that selects for variants of homoserine kinase that improve the efficiency of a novel biochemical pathway for synthesis of a critical cofactor. The Broader Impacts of this project will be to develop new approaches that are widely applicable to synthetic biology. One of the primary goals of this project is to make these techniques easy to apply to a broad spectrum of protein evolution applications by researchers with diverse molecular biology skills. Through this project, graduate and undergraduate students will be exposed to research and coursework at the Chemistry-Biology interface as developed collaboratively by Professors Batey and Copley.