Abstract

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.

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Accessions

Primary accessions

GenBank/EMBL/DDBJ

Data deposits

SynIXR and semi-synVIL sequences have been deposited to GenBank with the accession codes: synIXR, JN020955; semi-synVIL, JN020956. Microarray data have been submitted to Gene Expression Omnibus under accession number GSE31326.

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Acknowledgements

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.

Author information

Author notes

    • Jessica S. Dymond
    • , William J. Blake
    • , Joy W. Schwerzmann
    • , Junbiao Dai
    • , Derek L. Lindstrom
    •  & Annabel C. Boeke

    Present addresses: Animal Biosciences and Biotechnology Laboratory, Agricultural Research Service, USDA, 10300 Baltimore Avenue, Beltsville, Maryland 20705, USA (J.S.D.) . GreenLight Biosciences, Inc., 196 Boston Avenue, Suite 2400, Medford, Massachusetts 02155, USA (W.J.B.) ; Battelle Memorial Institute, 2987 Clairmont Road NE, Atlanta, Georgia 30329, USA (J.W.S.) ; School of Life Sciences, Tsinghua University, Beijing 100084, China (J.D.) ; Agilent Laboratories, 5301 Stevens Creek Boulevard, Mailstop 53L-IB, Santa Clara, California 95051, USA (D.L.L.); Bowdoin College, 5000 College Station, Brunswick, Maine 04011, USA (A.C.B.).

Affiliations

  1. High Throughput Biology Center, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, Maryland 21205, USA

    • Jessica S. Dymond
    • , Sarah M. Richardson
    • , Candice E. Coombes
    • , Timothy Babatz
    • , Junbiao Dai
    • , Annabel C. Boeke
    • , Joel S. Bader
    •  & Jef D. Boeke
  2. Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA

    • Jessica S. Dymond
    • , Candice E. Coombes
    • , Junbiao Dai
    •  & Jef D. Boeke
  3. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, Maryland 21205, USA

    • Sarah M. Richardson
    •  & Timothy Babatz
  4. Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205, USA

    • Héloïse Muller
    • , Narayana Annaluru
    • , Joy W. Schwerzmann
    •  & Srinivasan Chandrasegaran
  5. Codon Devices, 99 Erie Street, Cambridge, Massachusetts 02139, USA

    • William J. Blake
  6. Fred Hutchinson Cancer Research Center, Mailstop A3-025, PO Box 19024, Seattle, Washington 98109, USA

    • Derek L. Lindstrom
    •  & Daniel E. Gottschling
  7. Department of Biomedical Engineering, Whiting School of Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA

    • Joel S. Bader

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Contributions

J.S.D., S.M.R., S.C., J.S.B. and J.D.B. designed experiments. J.S.D., S.M.R., C.E.C., T.B., H.M., N.A., J.W.S., J.D. and A.C.B. performed experiments. W.J.B. built the synIXR chromosome. D.L.L. and D.E.G. generated the integrated CRE-EBD cassette. J.S.D., S.M.R., J.S.B. and J.D.B. analysed data and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jef D. Boeke.

Supplementary information

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  1. 1.

    Supplementary Information

    This file contains Supplementary Text 1-8, Supplementary References and Supplementary Figures 1-13 with legends.

  2. 2.

    Supplementary Tables

    This file contains Supplementary Tables 1-8.  This file was corrected on 22 September 2011 due to an error in one of the tables.

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DOI

https://doi.org/10.1038/nature10403

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