Design, Synthesis, and Testing Toward a 57-Codon Genome

genomes with functions not commonly found in nature. Here, we report computational

design, synthesis, and progress toward assembly of a 3.97-megabase, 57-codon Escherichia

coli genome in which all 62,214 instances of seven codons were replaced with synonymous

alternatives across all protein-coding genes. We have validated 63% of recoded genes

by individually testing 55 segments of 50 kilobases each.We observed that 91% 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. This work underscores the feasibility of rewriting genomes and establishes a

framework for large-scale design, assembly, troubleshooting, and phenotypic analysis of

synthetic organisms.