Modular protein switches

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Each of the thousands of different proteins in a cell has a unique function. These functions are often regulated by other molecules (signals). Some proteins are switches; a signal molecule binds to the protein and switches its function from an "off" to an "on" state (or vice versa). The switch could sense the presence of a pollutant, or of an infection, and trigger defense mechanisms. It could sense the presence of cancer cells and trigger a cell-killing response. The potential sensing and response capabilities are tremendous. Two modular platforms for creating switches will be developed during this project. One will detect specific proteins, and the other will control gene expression. Success in the research of this proposal will enable others to develop protein switches with applications in synthetic biology for biomanufacturing as well as in basic biological research. Rigorous training of undergraduates and graduate students will expand the biotechnology workforce.Protein switches recognize input signals such as ligand concentration and trigger an output signal such as enzyme activity or DNA binding. Directed evolution has generated switches with high affinity for their ligand and large changes in output. However, developing suitable selections or screens to identify switches remains challenging and often greatly hinders switch development. In this project, modular protein switch platforms for protein sensing and for control of gene expression will be developed. The protein-sensing platforms will utilize designed ankyrin-repeat proteins and will trigger enzyme activity or fluorescence. The platform for modular control of gene expression will utilize the nuclease-null dCas9 protein of the Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) system, and protein domains that bind inexpensive small molecules. The switches will function as inducible repressors of gene expression, with the small molecules serving as low-cost inducers. The development of these modular platforms will provide a set of functional switches for biotechnological use and for mechanistic study to facilitate the design of future switches. These switches will be used in basic scientific research to understand cellular function and applied research in synthetic biology and metabolic engineering.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.