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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, Xma I or Not I. ( 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. 

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, Xma I or Not I. ( 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. 

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Designer Chromosome One of the ultimate aims of synthetic biology is to build designer organisms from the ground up. Rapid advances in DNA synthesis has allowed the assembly of complete bacterial genomes. Eukaryotic organisms, with their generally much larger and more complex genomes, present an additional challenge to synthetic biologists. Annalur...

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... cerevisiae has a ge- nome size of ~12 Mb distributed among 16 chromosomes. The entire genome encodes ~6000 genes, of which ~5000 are individually nones- sential ( 1 ). Which of these nonessential genes are simultaneously dispensable? Although a number of studies have successfully mapped pairwise “synthet- ic lethal” interactions between gene knockouts, those methods do not scale well to three or more gene combina- tions because the number of combina- tions rises exponentially. Our approach to address this question is to produce a synthetic yeast genome with all nones- sential genes flanked by loxPsym sites to enable inducible evolution and ge- nome reduction (a process we refer to as SCRaMbLEing) in vivo ( 2 , 3 ). The availability of a fully synthetic S. cere- visiae genome will allow direct testing of evolutionary questions—such as the maximum number of nonessential genes that can be deleted without a catastrophic loss of fitness and the catalog of viable 3-gene, 4-gene, ... n - gene deletions that survive under a given growth condition—that are not otherwise easily approachable in a systematic unbiased fashion. Engineer- ing and synthesis of viral and bacterial genomes have been reported in the literature ( 4 – 11 ). An international group of scientists has embarked on constructing a designer eukaryotic genome, Sc2.0 (www.syntheticyeast.org), and here we report the total synthesis of a complete designer yeast chromosome. Yeast chromosome III, the third smallest in S. cerevisiae [316,617 base pairs (bp)], contains the MAT locus determining mating type and was the first chromosome sequenced ( 12 ) . We designed synIII according to fitness, genome stability, and genetic flexibility principles developed for the Sc2.0 genome ( 2 ). The native sequence was edited in silico by using a series of deletion, insertion, and base substitu- tion changes to produce the desired “designer” sequence (Fig. 1, figs. S1 and S2, and supplementary text). The hierarchical wet-laboratory workflow used to construct synIII (Fig. 2) con- sisted of three major steps: (i) The 750- bp building blocks (BBs) were pro- duced starting from overlapping 60- to 79-mer oligonucleotides and assembled by using standard polymerase chain reaction (PCR) methods ( 13 , 14 ) by undergraduate students in the ...

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