Reengineering the Genetic Code to Expand the Language of Biology Using a Cell-Free Synthetic Biology Approach

The incorporation of non-standard amino acids (nsAAs) is a protein engineering game changer that is transforming our ability to make better therapeutics, medical diagnostics, and biocatalysts. However, as we begin to explore the potential of incorporating more than one nsAA simultaneously into our protein of interest, we are limited by the genetic code.   While theoretically only 21 codons are necessary to incorporate the natural proteogenic cannon of 20 amino acids, all of the 64 natural codon possibilities have a corresponding tRNA or release factor.  Hence there is a need to reengineer the genetic code to emancipate codons for nsAA use beyond the popular amber stop codon target.  Here report the application of a cell-free synthetic biology approach toward this objective. 

A cell-free approach is well suited toward our application due to the direct access afforded by the system.  This access enables rapid optimization and unprecedented control of both the endogenous protein synthesis machinery and the exogenous machinery necessary for nsAA incorporation.  In addition, the cell-free system is not limited by the transport or toxicity of non-standard products and intermediates (e.g. nsAA, exogenous machinery necessary for nsAA incorporation).  Finally, as we consider transfer to commercial applications, the cell-free system has been demonstrated to be reliably and easily scaled from the laboratory scale to the commercial scale.