Genetic Code Compression and Expansion

Genetic code expansion enables the site-specific, incorporation of non-canonical amino acids (ncAAs) into proteins, and has enabled diverse biological discoveries. Most experiments incorporate ncAAs in response to amber stop codons. This strategy is limited to incorporating one type of ncAAs into a protein at a time, and there are no other natural blank codons that can be used for ncAA incorporation. We synthesized a 4 Mb E. coli genome with a compressed genetic code, through the genome-wide substitution of three target codons by defined synonyms. The resulting cell, Syn61, uses 61 codons to encode the 20 canonical amino acids. In Aim 1 we address the limitations of genetic code expansion by encoding multiple distinct ncAAs in response to blank codons in Syn61. This dramatically expands the applications of ncAA incorporation. The recoding of Syn61 was based on the prior identification of a synonymous codon compression scheme (a rule that defines which codons to remove and which codons to replace them with). In Aim 2 and Aim 3 we systematically discover allowed synonymous codon compression schemes that we use to create deeply compressed synthetic genomes using accelerated genome synthesis methods. This creates more blank codons that may be reassigned to ncAAs.