Figure 1. Design of a synthetic 57-codon Escherichia coli genome.

(a) The design of Ec_Syn57’s compressed genetic code. Magenta marks codons and their corresponding tRNA genes and release factor I (RF1, encoded by prfA) selected for elimination during genome design. (b) The computational genome design of Ec_Syn57 synonymously recoded all known instances of the seven target codons highlighted in Figure 1a and streamlined the synthetic chromosome. Prior to synonymous recoding, (I.) overlapping genes that contain forbidden codons within their overlapping region were disentangled while preserving the downstream gene’s ribosomal binding site; next, (II.) protein-coding genes were recoded to confer the 57-codon genetic code of Figure 1a while minimizing local changes in GC% and mRNA folding differences near the 5’ end of genes. Next, we streamlined DNA synthesis and subsequent genome assembly steps by eliminating unstable repeats (III.), removing cut sites of AarI, BsaI, and BsmBI restriction enzymes (IV.), and eliminating sequences containing >8 consecutive As, Cs, Ts, or >5 consecutive Gs (V.). Finally, the refactored, recoded genome was divided into 86 ~50 kbp segments, and the entire genome was synthesized.