Rewriting the Proteomic Alphabet in Cell-Free Systems

The vast majority of biology relies on just 20 canonical amino acids to make the millions of diverse proteins that help create life. The addition of non-canonical amino acids (ncAA) to this amino acid “alphabet” has proven a promising tool in for biochemistry and microbiology with applications such as residue-specific NMR, site-specific conjugation, and enzyme engineering. However, current biological methods for incorporating ncAA are commonly limited by transport of the ncAA into the cells, difficulty optimizing the concentration/activity of necessary exogenous machinery, and competition from native machinery (e.g. release factors when targeting amber stop codons).  We present advances in cell-free transcription/translation technology to overcome these limitations and also provide cost and yield analysis of cell-free systems directly compared to state-of-the-art in vivo systems.