This project is jointly funded by the Chemistry of Life Processes Program in the Division of Chemistry in the Directorate of Mathematical and Physical Sciences and the Systems and Synthetic Biology Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences. Proteins are the central actors in living systems, performing the vast majority of catalytic, signaling, structural, and controlling functions in cells. The chemistry of proteins is largely determined by the chemistry of the 20 genetically-encoded amino acid building blocks of proteins. Although sufficient for living systems, this set of natural amino acids encodes a limited set of chemical functionalities. The expansion of the genetically encodable chemical functionality beyond that afforded by the 20 natural amino acids will open vast new vistas of protein sequence and functional space for exploration and exploitation. Enlarging the encodable chemistry of proteins through genetic code expansion is an enabling technology that offers the potential to dramatically increase the functions of proteins. This research will explore the space of sense codon reassignment in E. coli to identify a set of reassignable codons for genetic code expansion. The research will lead to improved technologies for non-canonical amino acid incorporation that allow multiple copies of multiple non-canonical amino acids to be incorporated into any protein of interest. The pursuit of these experiments will help prepare of the next generation of interdisciplinary scientists and engineers to contribute to a strong competitive STEM workforce. The legacy infrastructure built in the form of tRNA/aminoacyl tRNA synthetase systems optimized for sense codon reassignment will provide new tools to the protein science research community and direct benefits to society through rapid increases in the rate of generation of improved protein-based materials and devices.
Under this award, Fisk and his research team will study the expansion of the genetic code through sense codon reassignment. The research project will employ a fluorescence-based screen to measure the extent to which sense codons that naturally read through wobble interactions in E. coli can be reassigned using the orthogonal tRNA/aaRS pairs most commonly employed for nonsense suppression. The same fluorescence screen will be further employed to evolve the tRNA/aaRS systems to better decode reassigned sense codons, to evaluate the plasticity of aminoacyl tRNA synthetase-tRNA recognition domains, and explore the extent to which the machinery of translation can be molded to accommodate expanded genetic codes. The systems identified and optimized to reassign sense codons will be combined to generate genetic codes containing 22 and 23 amino acids. The combined experimental aims of the project provide a host of new measurements to better map the plasticity of the translational system in E. coli and evaluate the degree to which representative tRNA/aaRS pairs can be modified to reassign the meaning of sense codons.
