The genetic code of all known organisms is comprised of the 20 proteinogenic amino acids that serve as building blocks on a peptide chain to form a vast array of proteins. Proteins are responsible for virtually every biological process in all organisms; however, the 20 amino acids contain a limited number of functional groups that often leaves much to be desired. The lack of diversity addresses the need to increase the genetic repertoire of living cells to include a variety of amino acids with novel structural, chemical, and physical properties not found in the common 20 amino acids. In order to expand the chemical scope of the genetic code beyond the functionalities that can be directly genetically encoded, unnatural amino acids must be added to the proteome. The ability to incorporate unnatural amino acids (UAAs) into proteins at defined sites has a direct impact on the ability of scientists to study biological processes that are difficult or impossible to address by more classical methods. The UUAs of interest are acylated lysine derivatives (isovaleryl, isobutyryl, and β-hydroxybutyryl) and photocaged histidine. Acylation of histone lysine has been linked to epigenetic regulation of metabolism.1 A means to site-specifically incorporate each acylated lysine derivative would help study the effect of acylated lysine in epigenetic regulation. Likewise, in order to elucidate the role of histidine in specific protein functions, one can replace a critical histidine with a photocaged histidine. Photocaged amino acids are those that possess a photo-cleavable, aromatic caged group. Light-induced protein activation allows for the biological activity of the protein to be spatiotemporally regulated under non-invasive external control.2 The site-specific in vivo incorporation of unnatural amino acids is made possible by amber codon suppression by an orthogonal suppressor aminoacyl-tRNA synthetase (aaRS)/tRNA pair.3 In amber codon suppression the amber stop codon is decoded for an UAA by a suppressor aaRS/tRNA pair. To accept the UAA, the aaRS must be evolved to achieve orthogonal activity with specific UUAs. The pyrrolysyl aaRS/tRNA (PylRS/PylT) pair from M. barkeri and M. mazei was used to construct multiple, large-scale aaRS mutant libraries where critical residues within the active site of PylRS are mutated via site-saturated mutagenesis.4 The libraries were subjected to directed evolution through a series of positive and negative selections to enrich aaRS variants that exclusively bind to acylated lysine derivatives and photocaged histidine as substrates.5 The PylRS selection survivors were screened for UAA activity and identified successful clones underwent a fluorescent activity assay. The active aaRS were used for amber codon suppression to express the respective UAA in ubiquitin and green fluorescent protein constructs.