(734d) Rewriting the Language of Biology: Unnatural Amino Acids Incorporation at Sense Codons Using Cell-Free System

Authors: 
Salehi, S. M. A., Brigham Young University
Smith, M. T., Brigham Young University
Bundy, B. C., Brigham Young University
Schinn, S. M., University of California, San Diego
Hunt, J., Stanford University

Although 20 canonical amino acids are life’s building blocks, today’s applications (e.g. biocatalysts, biotheraputics, site–specific conjugation) often require unique chemical functionality beyond those amino acids. Therefore, site-specific incorporation of multiple unnatural amino acids (uNAA) has been a major aspiration of protein engineering and synthetic biology. While there are many promising efforts to incorporate uNAA, the amber stop codon is the predominant coding target, due to the fundamental constraints imposed by the genetic code. In theory, the great redundancy in genetic code has potential to be exploited for uNAA, but such extensive rewriting is extremely difficult in biological systems.

To address this challenge, we propose a cell-free synthetic biology approach to rewrite the genetic code and incorporate multiple uNAAs simultaneously. The open nature of the cell-free system enables us to modify the system and reengineer the genetic code to emancipate some codons for incorporating uNAAs. In fact, there is potential to emancipate up to 43 codons, since only 21 of 64 codons are required for translation of 20 standard amino acids and translation termination. To that end, we removed all E. coli endogenous tRNA and then repopulated the system with a designed minimal set of synthetic tRNA made in vitro. By importing orthogonal sets of tRNA/synthetase from different organisms, we were able to emancipate codons and demonstrate the incorporation of uNAA at sense codons.