Fig. 2: Construction of synthetic genomes.

Synthetic oligonucleotides are assembled into progressively larger double-stranded DNA fragments using methods such as polymerase cycling assembly (PCA), digestion and ligation, and transformation-associated recombination (TAR) in yeast. a The synthetic Mycoplasma mycoides genome was assembled via TAR in yeast from 1 kb DNA cassettes gradually into larger fragments (10 kb and 100 kb), and finally into 1.08 Mb genome, prior to extraction and genome transplantation into M. capricolum to generate a new M. mycoides strain controlled by the synthetic genome (JCVI-syn1.0). b Genome-wide recoding was conducted to generate an E. coli strain with 61 codons. The recoded genome was assembled from 10 kb fragments into 100 kb fragments on a bacterial artificial chromosome via TAR in yeast. A 100 kb fragment was integrated into different E. coli strains by replicon excision for enhanced genome engineering through programmed recombination (REXER), and 4–5 100 kb fragments in total were assembled as a big section by genome stepwise interchange synthesis (GENESIS). These large DNA sections were assembled into a whole recoded genome through a conjugation-based strategy, which generated Syn61. c The Sc2.0 was initially constructed from 750 bp building blocks, and through PCA and TAR in yeast into 2–4 kb minichunks; or in more-recent Sc2.0 chromosomes, assembled from 6–10 kb chunks into 30–50 kb megachunks via in vitro digestion and ligation. The minichunks or megachunks were then transformed into yeast cell to replace the native genome iteratively.