Encoded Cellular Synthesis of Unnatural Biopolymers

Final Report Summary - ECSUB (Encoded Cellular Synthesis of Unnatural Biopolymers)

1) We developed new systems for incorporating unnatural amino acids into proteins. 2) We showed that these could be used in a range of hosts drastically expanding the scope of genetic code expansion and unnatural amino acid mutagenesis. 3) We showed that a range of useful unnatural amino acids could be incorporated using the new pair- including key post translational modifications (ubiquitination, acetylation, methylation), 4) We developed approaches for rapidly and site specifically labeling proteins inside cells with diverse fluorophores for superresolution imaging, 5) We developed approaches to control protein function with light via the installation of photocaged amino acids. We applied the approaches we developed to provide previously unattainable insight into: i) the role of post translational modifications in regulating structure, enzymology, biological function, ii) the kinetics of signaling and transport, iii) the properties of proteins in cells, 6) We demonstrated that the orthogonal ribosome can be evolved to read quadruplet codons, 7) We demonstrated two routes to creating mutually orthogonal synthetase/tRNA pair, 8) We demonstrated the incorporation of diverse sets of unnatural amino acid in a single polypeptide expressed in cells, 10) We demonstrated the incorporation of pairs of fluorophores for FRET to study protein conformational change. In addition to the discoveries we have made with the tools we have created, the materials for the approaches we have developed have been extensively distributed, and used by others to make new discoveries.

Final Report Summary - ECSUB (Encoded Cellular Synthesis of Unnatural Biopolymers)

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