Incorporation of non-standard amino acids into proteins: challenges, recent achievements, and emerging applications Biological Engineering, Biotechnology The natural genetic code only allows for 20 standard amino acids in protein translation, but genetic code reprogramming enables the incorporation of non-standard amino acids (NSAAs). Proteins containing NSAAs provide enhanced or novel properties and open diverse applications. With increased attention to the recent advancements in synthetic biology, various improved and novel methods have been developed to incorporate single and multiple distinct NSAAs into proteins. However, various challenges remain in regard to NSAA incorporation, such as low yield and misincorporation. In this review, we summarize the recent efforts to improve NSAA incorporation by utilizing orthogonal translational system optimization, cell-free protein synthesis, genomically recoded organisms, artificial codon boxes, quadruplet codons, and orthogonal ribosomes, before closing with a discussion of the emerging applications of NSAA incorporation. Sponsorship: NIH R15AI130988 Jin, Xing Park Oh-Jin Hong, Seok Hoon 2019 Article islandora:1012024 http://hdl.handle.net/10560/islandora:1012024 ChBE / Chemical and Biological Engineering Illinois Institute of Technology en Open Access