Recent advances in DNA synthesis technology have enabled the construction of novel genetic pathways and genomic elements, furthering our understanding of system-level phenomena1,2,3,4,5,6,7. The ability to synthesize large segments of DNA allows the engineering of pathways and genomes according to arbitrary sets of design principles. Here we describe a synthetic yeast genome project, Sc2.0, and the first partially synthetic eukaryotic chromosomes, Saccharomyces cerevisiae chromosome synIXR, and semi-synVIL. We defined three design principles for a synthetic genome as follows: first, it should result in a (near) wild-type phenotype and fitness; second, it should lack destabilizing elements such as tRNA genes or transposons8,9; and third, it should have genetic flexibility to facilitate future studies. The synthetic genome features several systemic modifications complying with the design principles, including an inducible evolution system, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution). We show the utility of SCRaMbLE as a novel method of combinatorial mutagenesis, capable of generating complex genotypes and a broad variety of phenotypes. When complete, the fully synthetic genome will allow massive restructuring of the yeast genome, and may open the door to a new type of combinatorial genetics based entirely on variations in gene content and copy number.
We thank G. Church for suggesting the global substitution of TAG codons with TAA codons, C. Connelly for sharing technical expertise and V. Huang for generating a sequence visualizer. We are grateful to B. Cormack, G. Seydoux and J. Nathans for offering helpful advice, to Y. Cai and J. Peccoud for suggesting methods to validate the sequence data, and to E. Louis for providing expert advice on telomeres. The work was supported by National Science Foundation grant MCB0718846 to J.D.B., J.S.B. and S.C.; by a grant from Microsoft to J.S.B. and J.D.B.; by Department of Energy Fellowship DE-FG02097ER25308 to S.M.R.; by National Institutes of Health grant AG023779 to D.E.G.; and by a fellowship from Fondation pour la Recherche Médicale to H.M.