Research Article

Total Synthesis of a Functional Designer Eukaryotic Chromosome

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Science  04 Apr 2014:
Vol. 344, Issue 6179, pp. 55-58
DOI: 10.1126/science.1249252

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  1. Fig. 1 SynIII design.

    Representative synIII design segments for loxPsym site insertion (A and B) and stop codon TAG to TAA editing (C) are shown. Green diamonds represent loxPsym sites embedded in the 3′ untranslated region (UTR) of nonessential genes and at several other landmarks. Fuchsia circles indicate synthetic stop codons (TAG recoded to TAA). Complete maps of designed synIII chromosome with common and systematic open reading frame (ORF) names, respectively, are shown in figs. S1 and S2.

  2. Fig. 2 SynIII construction.

    (A) BB synthesis. JHU students in the Build-A-Genome course synthesized 750-bp BBs (purple) from oligonucleotides. nt, nucleotides. (B) Assembly of minichunks. Two- to 4-kb minichunks (yellow) were assembled by homologous recombination in S. cerevisiae (table S1). Adjacent minichunks were designed to encode overlap of one BB to facilitate downstream assembly steps. Minichunks were flanked by a rare cutting restriction enzyme (RE) site, XmaI or NotI. (C) Direct replacement of native yeast chromosome III with pools of synthetic minichunks. Eleven iterative one-step assemblies and replacements of native genomic segments of yeast chromosome III were carried out by using pools of overlapping synthetic DNA minichunks (table S2), encoding alternating genetic markers (LEU2 or URA3), which enabled complete replacement of native III with synIII in yeast.

  3. Fig. 3 Characterization and testing of synIII strain.

    (A) PCRTag analysis (one PCRTag per ~10 kb) of the left arm of synIII and WT yeast (BY4742) DNA is shown. Analysis of the complete set of PCRTags is shown in figs. S4 to S6. (B) Karyotypic analysis of synIII and synIIIL strains by pulsed-field gel electrophoresis revealed the size reduction of synIII and synIIIL compared with native III. Yeast chromosome numbers are indicated on the right side. SynIII (272,871 bp) and native chromosome VI (270,148 bp) comigrate in the gel. A karyotypic analysis of synIII and all intermediate strains is shown in fig. S8. (C) SynIII and synIIIL phenotyping on various types of media. Tenfold serial dilutions of saturated cultures of WT (BY4742), synIIIL, and synIII strains were plated on the indicated media and temperatures. YPD, yeast extract peptone dextrose; YPGE, yeast extract peptone glycerol ethanol; MMS, methyl methanosulfate. A complete set of synIII and synIIIL phenotyping under various conditions is shown in fig. S11.

  4. Fig. 4 Genomic stability of the synIII strain.

    (A) PCRTag analysis of synIII strain after ~125 generations. We assayed for the loss of 58 different segments lacking essential genes in the absence of SCRaMbLEing; no losses were observed after over 200,000 segment-generations analyzed; reported frequency is a maximum estimate of segment loss frequency per generation. gDNA, genomic DNA. (B) Evaluation of the loss rate of synIII chromosome using a-like faker assay. No significant change in the loss frequency was observed, although the absolute loss rate value is modestly higher in synIII. SD, standard dextrose. (C) SCRaMbLE leads to a gain of mating type a behavior in synIII heterozygous diploids. Frequencies are of a-mater and α-mater colonies post-SCRaMbLE (induction with estradiol) in synIII/III and III/III strains. A complete SCRaMbLE analysis is shown in fig. S18. Diamonds represent loxPsym sites, and circles indicate centromeres.