Synthetic chromosome arms function in yeast and generate phenotypic diversity by design

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Maps of synIXR and semi-synVIL.
Figure 2: Strain construction and verification.
Figure 3: Transcript profiling of wild-type and synIXR strains.
Figure 4: SCRaMbLE rearranges genomes.

Accession codes

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.

References

  1. 1

    Han, J. S. & Boeke, J. D. A highly active synthetic mammalian retrotransposon. Nature 429, 314–318 (2004)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Richardson, S. M., Wheelan, S. J., Yarrington, R. M. & Boeke, J. D. GeneDesign: rapid, automated design of multikilobase synthetic genes. Genome Res. 16, 550–556 (2006)

    CAS  Article  Google Scholar 

  3. 3

    Chan, L. Y., Kosuri, S. & Endy, D. Refactoring bacteriophage T7. Mol. Syst. Biol. 1, 2005.0018 (2005)

    Article  Google Scholar 

  4. 4

    Stricker, J. et al. A fast, robust and tunable synthetic gene oscillator. Nature 456, 516–519 (2008)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Gibson, D. G. et al. One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. Proc. Natl Acad. Sci. USA 105, 20404–20409 (2008)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Gibson, D. G. et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52–56 (2010)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Lartigue, C. et al. Creating bacterial strains from genomes that have been cloned and engineered in yeast. Science 325, 1693–1696 (2009)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Ji, H. et al. Hotspots for unselected Ty1 transposition events on yeast chromosome III are near tRNA genes and LTR sequences. Cell 73, 1007–1018 (1993)

    CAS  Article  Google Scholar 

  9. 9

    Admire, A. et al. Cycles of chromosome instability are associated with a fragile site and are increased by defects in DNA replication and checkpoint controls in yeast. Genes Dev. 20, 159–173 (2006)

    CAS  Article  Google Scholar 

  10. 10

    Churcher, C. et al. The nucleotide sequence of Saccharomyces cerevisiae chromosome IX. Nature 387, 84–87 (1997)

    CAS  PubMed  Google Scholar 

  11. 11

    Park, H. et al. Expanding the genetic code of Escherichia coli with phosphoserine. Science 333, 1151–1154 (2011)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Isaacs, F. J. et al. Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science 333, 348–353 (2011)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Hoess, R. H., Wierzbicki, A. & Abremski, K. The role of the loxP spacer region in P1 site-specific recombination. Nucleic Acids Res. 14, 2287–2300 (1986)

    CAS  Article  Google Scholar 

  14. 14

    Vollrath, D., Davis, R. W., Connelly, C. & Hieter, P. Physical mapping of large DNA by chromosome fragmentation. Proc. Natl Acad. Sci. USA 85, 6027–6031 (1988)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Lindstrom, D. L. & Gottschling, D. E. The mother enrichment program: a genetic system for facile replicative life span analysis in Saccharomyces cerevisiae. Genetics 183, 413–422 (2009)

    CAS  Article  Google Scholar 

  16. 16

    Kuras, L., Cherest, H., Surdin-Kerjan, Y. & Thomas, D. A heteromeric complex containing the centromere binding factor 1 and two basic leucine zipper factors, Met4 and Met28, mediates the transcription activation of yeast sulfur metabolism. EMBO J. 15, 2519–2529 (1996)

    CAS  Article  Google Scholar 

  17. 17

    Ogawa, H. & Fujioka, M. Purification and characterization of saccharopine dehydrogenase from baker’s yeast. J. Biol. Chem. 253, 3666–3670 (1978)

    CAS  PubMed  Google Scholar 

  18. 18

    Sambrook, J. & Russell, D. W. Isolation of BAC DNA from small-scale cultures. Cold Spring Harb. Protoc. 10.1101/pdb.prot4006 (2006)

  19. 19

    Hoffman, C. S. Preparation of yeast DNA. Curr. Protoc. Mol. Biol. Ch. 13, Unit 13.11. (2001)

  20. 20

    Boeke, J. D., Garfinkel, D. J., Styles, C. A. & Fink, G. R. Ty elements transpose through an RNA intermediate. Cell 40, 491–500 (1985)

    CAS  Article  Google Scholar 

  21. 21

    Schwartz, D. C. & Cantor, C. R. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37, 67–75 (1984)

    CAS  Article  Google Scholar 

  22. 22

    Lemoine, F. J., Degtyareva, N. P., Lobachev, K. & Petes, T. D. Chromosomal translocations in yeast induced by low levels of DNA polymerase: a model for chromosome fragile sites. Cell 120, 587–598 (2005)

    CAS  Article  Google Scholar 

  23. 23

    Louis, E. J. The chromosome ends of Saccharomyces cerevisiae. Yeast 11, 1553–1573 (1995)

    CAS  Article  Google Scholar 

  24. 24

    Parenteau, J. et al. Deletion of many yeast introns reveals a minority of genes that require splicing for function. Mol. Biol. Cell 19, 1932–1941 (2008)

    CAS  Article  Google Scholar 

  25. 25

    Percudani, R., Pavesi, A. & Ottonello, S. Transfer RNA gene redundancy and translational selection in Saccharomyces cerevisiae. J. Mol. Biol. 268, 322–330 (1997)

    CAS  Article  Google Scholar 

  26. 26

    Dymond, J. S. et al. Teaching synthetic biology, bioinformatics and engineering to undergraduates: the interdisciplinary build-a-genome course. Genetics 181, 13–21 (2009)

    CAS  Article  Google Scholar 

  27. 27

    Hampsey, M. A review of phenotypes in Saccharomyces cerevisiae. Yeast 13, 1099–1133 (1997)

    CAS  Article  Google Scholar 

  28. 28

    Abramoff, M. D., Magelhaes, P. J. & Ram, S. J. Image processing with ImageJ. Biophotonics Int. 11, 36–42 (2004)

    Google Scholar 

  29. 29

    Blake, W. J. et al. Pairwise selection assembly for sequence-independent construction of long-length DNA. Nucleic Acids Res. 38, 2594–2602 (2010)

    CAS  Article  Google Scholar 

Download references

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

Affiliations

Authors

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.

Corresponding author

Correspondence to Jef D. Boeke.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Text 1-8, Supplementary References and Supplementary Figures 1-13 with legends. (PDF 1729 kb)

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. (PDF 908 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Dymond, J., Richardson, S., Coombes, C. et al. Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature 477, 471–476 (2011). https://doi.org/10.1038/nature10403

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.