We will build an orthogonal translation system for the encoded synthesis of unnatural polymers in cells. The steps towards this goal are each significant and substantial and, importantly, our solutions for each step are both scalable and modular with those for other steps in the process. In the first step we create a scalable and modular route to multiple new aminoacyl-tRNA synthetase/tRNA pairs that are orthogonal to each other and to endogenous synthetases and tRNAs. In a second step we engineer a cell for orthogonal translation to enable each orthogonal tRNA to quantitatively and efficiently decode a distinct quadruplet codon. In a third step we invent a modular and scalable route to program the active sites of mutually orthogonal synthetases to uniquely recognize distinct unnatural building blocks. The modular combination of the advances in each step enables the conversion of genetic code expansion with one or two unnatural amino acids into a scalable technology for genetic code reprogramming, facilitating the encoded synthesis of unnatural polymers using a synthetic genetic code with more encoded monomers than the natural genetic code.
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