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(129e) Rewriting the Proteomic Alphabet in Cell-Free Systems

Smith, M. T., Brigham Young University
Bundy, B. C., Brigham Young University
Hunt, J., Stanford University

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.