Research Article

Design of a synthetic yeast genome

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Science  10 Mar 2017:
Vol. 355, Issue 6329, pp. 1040-1044
DOI: 10.1126/science.aaf4557
  • Fig. 1 Sc2.0 Consortium chromosome assignments.

    The chromosomes (roman numerals) and their approximate sizes are indicated, along with the lead institution(s) involved in performing chromosome synthesis, assembly, and debugging, as well as raising the needed funds. The gel on the right shows a typical pulsed-field gel of a wild-type strain, with the chromosome-sized DNA molecules identified. Black dots indicate chromosome centromeres. BGI, Beijing Genomics Institute; JGI, Joint Genome Institute of the U.S. Department of Energy; JHU, The Johns Hopkins University; NYU, New York University; U, University; Res Inst, Research Institute.

  • Fig. 2 Megachunk assembly.

    (A) Megachunks are assembled from three to six chunks by RE (restriction enzyme) cutting and ligation. RE sites have distinct nonpalindromic overhangs to assure proper assembly. (B) Sequential SwAP-In HR steps to complete synthetic chromosome (bottom). Colored segments, megachunks; black, native sequence; blue and red triangles, LEU2 and URA3, respectively; X, sites of HR; 5-FOAS, 5-fluoroorotic acid (FOA)–sensitive allele. Inserts between dashed lines are UTCs (universal telomere caps). (C) Detail of right end of each megachunk. Each chunk begins and ends with a RE site (scissors) designed to ligate specifically to the adjacent chunk(s). However, the rightmost chunk in each megachunk contains three types of sequence: synthetic (colors), marker (colored triangle), and native (black). The marker is inserted in an endogenous nonessential gene during assembly of each megachunk.

  • Fig. 3 Consolidation of Sc2.0 chromosomes by endoreduplication intercross.

    (A) A pGAL-CENx construct was integrated into the native chromosome in pairs of synthetic chromosome strains, which were then mated. After growth on galactose to induce destabilization of native chromosomes and selection on FOA medium, the 2n – 2 state was confirmed by PCRTag analysis. Haploid poly-syn chromosome strains were then generated by sporulation and dissection. An episomal copy of MATa was introduced to permit sporulation in synIII-containing strains. Double-syn strains were generated similarly (fig. S6). (B) Electrophoretic karyotypes of syn strains showing faster migration of synIII and synVI compared with native strains; note that IXL-synIXR and native IX migrate identically.

  • Table 1 Design challenges and policies adopted.

    CDS, gene coding sequence; snoRNA, small nucleolar RNA.

    Design challenge or amendmentPolicy adopted by design team
    Subtelomeric repeats
    of varying copy number
    on multiple chromosomes
    Delete and monitor for phenotypes
    as chromosomes are combined. Exception:
    vitamin biosynthesis genes retain one copy.
    Dispersed repeated genes of high copy
    number, as well as high-copy COS and
    seripauperin genes
    Delete and monitor for phenotypes as
    chromosomes are combined.
    loxPsym sites <300 bp apart when
    inserted algorithmically (not especially
    useful and more difficult to synthesize)
    loxPsym thinning to
    eliminate the loxPsym site
    closer to the centromere.
    Stop codon overlaps a second CDS;
    insertion of loxPsym site would disrupt
    second CDS; also TAG recoding to TAA
    could disrupt CDS
    Favor preservation of “verified ORFs”
    over “dubious ORFs” and “uncharacterized ORFs”;
    always add loxPsym site to a verified ORF in this case
    Tandem repeats inside CDSs (34)Use GeneDesign’s RepeatSmasher
    module to recode such genes
    to minimize DNA level repetitiveness,
    making DNA easier to synthesize and assemble.
    Homopolymer tracts, including frequent
    A and T tracts, are difficult to synthesize
    In synthesis phase, permit 10% length
    variation for homopolymer
    tracts >10 bp provided they are
    in a noncoding region.
    IntronsDelete pre-mRNA introns precisely, except from genes
    with evidence of a fitness defect caused by intron
    deletion (35, 36). The HAC1 intron, which uses separate
    splicing machinery and is known to play a critical
    role in regulation of the unfolded protein response,
    was not deleted (9). Delete all tRNA introns precisely.
    Intronically embedded snoRNAsThese are individually nonessential and
    were deleted with their host introns.
    They could be “refactored” by
    insertion into the array of snoRNAs on chr II.
  • Table 2 Versions of synV.

    These designed versions (12) include those created by computational specialists (CS), yeast specialists (YS), and live strains synthesized (Syn).

    VersionDescriptionWorkflow or associated strain(s)
    5_0_00Wild type
    5_1_00PCRTags addedBioStudio PCR tagger (CS)
    5_2_00Stop codons swappedBioStudio codon juggler (CS)
    5_3_00loxP sites insertedBioStudio chromosome splicer (CS)
    5_3_01 ~ 5_3_38Intermediate editing stepsBioStudio graphical user interface (YS)
    5_3_39SegmentationBioStudio chromosome splitter (YS)
    5_3_40 ~ 5_3_43Intermediate sequence/
    annotation corrections
    BioStudio reports and expert review (CS, YS)
    5_9_01 ~ 5_9_21Draft synV strains with mutations
    compared to the final design
    version 5_3_43
    yXZX345, yXZX446, yXZX473, yXZX512,
    yXZX538, yXZX633, yXZX651, yXZX682,
    yXZX699, yXZX702, yXZX713, yXZX723,
    yXZX732, yXZX743, yXZX757, yXZX762,
    yXZX780, yXZX796, yXZX809,
    yXZX822, yXZX829 (Syn)
    5_9_22Final synV strain, same
    as designed
    yXZX846 (Syn)
  • Table 3 Summary statistics for design of Sc2.0.

    WT, wild type; SYN, synthetic.

    WT sizeSYN
    size
    No. of
    stop
    codon
    swaps
    No. of
    loxP
    sites
    added
    bp of
    PCRTag
    recorded
    bp of
    RE
    sites
    recoded
    No. of
    tRNA
    deleted
    bp of
    tRNA
    deleted
    bp of
    repeats
    deleted
    chr012302081810301962353521043723987
    chr0281318477003593271136511215139937030
    chr03316617272195441005272250107947358
    chr041531933145467118347925398229828226111674
    chr0557687453602461174876081320147111181
    chr0627014824274530694553369108359297
    chr071090940102895212638017910157236288713284
    chr085626435067056118699807141187819019
    chr094398854055135414279434361073611632
    chr10745751707459852491258211022418537523
    chr11666816659617681991176910171512434214
    chr12107817799940612229115129153919164610843
    chr139244318837491003371591102116917673
    chr14784333753096962601332911131411525115
    chr15109129110483431473991801520582016129542
    chr1694806690299412733415493137417133810048
    Total1207129711352534141639321992301608027221762149420

Supplementary Materials

  • Design of a synthetic yeast genome

    Sarah M. Richardson, Leslie A. Mitchell, Giovanni Stracquadanio, Kun Yang, Jessica S. Dymond, James E. DiCarlo, Dongwon Lee, Cheng Lai Victor Huang, Srinivasan Chandrasegaran, Yizhi Cai, Jef D. Boeke, Joel S. Bader

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Figs. S1 to S7
    • Movie S1 caption
    • References

    Images, Video, and Other Media

    Movie S1
    Movie depicting the editing process for synV
    Correction (20 March 2017): In this revision, the figures and references have been inserted. The conclusions of the Research Article remain unchanged.
    The original version is accessible here.