Design, Synthesis and Testing Towards a 57-Codon Genome

Recoding, the re-purposing of genetic codons, is a powerful strategy for enhancing genomes with functions not commonly found in nature. We report the computational design, synthesis, and progress towards assembly of a 3.97 MB, 57-codon Escherichia coli genome in which all 62,214 instances of seven codons were replaced with synonymous alternatives across all protein coding genes. Thus far, we have validated 63% of recoded genes by individually testing 55 segments of 50-kb each. Importantly, 90% of tested essential genes retained functionality with limited fitness effect. We demonstrate identification and correction of lethal design exceptions, only 13 of which were found in 2229 genes tested. This work underscores the feasibility of rewriting genomes and establishes a framework for large-scale design, assembly, troubleshooting and phenotypic analysis of synthetic organisms.