To create proteins, nature uses a set of 20 amino acids (called proteinogenic amino acids, or prAAs) throughout all of life. Even with this limited toolkit, a wide variety of functions can be achieved. However, chemical and molecular biologists have developed techniques for expanding this set of 20 amino acids to enable incorporation of hundreds of non-canonical amino acids (ncAAs). This feat is achieved by augmenting nature’s natural system for protein generation. In the first steps of protein synthesis, an aminoacyl-tRNA synthetase (aaRS) enzyme binds to a prAA and its partner tRNA. The aaRS catalyzes the covalent attachment of the prAA to its partner tRNA (aminoacylation or charging). The prAA-tRNA is trafficked to the ribosome, where it is used to decode mRNA to create a protein. The anticodon of the tRNA can hydrogen bond to specific codons in the mRNA, which links the mRNA sequence to the final protein sequence. For ncAA incorporation, we evolve an orthogonal aaRS/tRNA pair (o-aaRS and o-tRNA) to recognize a target ncAA and decode an infrequently used codon (typically the amber stop codon).