Whole genome synthesis: from poliovirus to synthetic yeast

Dai, Junbiao; Cai, Yizhi; Yuan, Yi; Yang, Huanming; Boeke, Jef D.

doi:10.1007/s40484-017-0101-x

Quant. Biol.

2017

DOI: 10.1007/s40484-017-0101-x

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Whole genome synthesis: from poliovirus to synthetic yeast

Junbiao Dai

Yizhi Cai

Yi Yuan

et al.

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Cited by 3 publications

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References 49 publications

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“…Saccharomyces cerevisiae was the first eukaryotic organism to have its genome sequenced, and it has become one of the primary targets of genome synthesis. − The Synthetic Yeast Genome Project (Sc2.0) aims to build a designer genome that will allow scientists to answer a series of important biological questions and facilitate its industrial applications. Each synthetic chromosome was segmented into different numbers of megachunks that could be integrated into the host genome, replacing the corresponding wild-type sequences by iterative homologous recombination, a method called switching auxotrophies progressively for integration (SwAP-In).…”

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confidence: 99%

Improving Chromosome Synthesis with a Semiquantitative Phenotypic Assay and Refined Assembly Strategy

et al. 2019

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Recent advances in DNA synthesis technology have made it possible to rewrite the entire genome of an organism. The major hurdles in this process are efficiently identifying and fixing the defect-inducing sequences (or "bugs") during rewriting. Here, we describe a highthroughput, semiquantitative phenotype assay for evaluating the fitness of synthetic yeast and identifying potential bugs. Growth curves were measured under a carefully chosen set of testing conditions. Statistical analysis revealed strains with subtle defects relative to the wild type, which were targeted for debugging. The effectiveness of the assay was demonstrated by phenotypic profiling of all intermediate synthetic strains of the synthetic yeast chromosome XII. Subsequently, the assay was applied during the process of constructing another synthetic chromosome. Furthermore, we designed an efficient chromosome assembly strategy that integrates iterative megachunk construction with CRISPR/Cas9-mediated assembly of synthetic segments. Together, the semiquantitative assay and refined assembly strategy could greatly facilitate synthetic genomics projects by improving efficiency during both debugging and construction.

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Improving Chromosome Synthesis with a Semiquantitative Phenotypic Assay and Refined Assembly Strategy

et al. 2019

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Whole genome engineering by synthesis

Luo

Yang

Geng

et al. 2018

Sci. China Life Sci.

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Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods

et al. 2018

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Exogenous pathway optimization and chassis engineering are two crucial methods for heterologous pathway expression. The two methods are normally carried out step-wise and in a trial-and-error manner. Here we report a recombinase-based combinatorial method (termed “SCRaMbLE-in”) to tackle both challenges simultaneously. SCRaMbLE-in includes an in vitro recombinase toolkit to rapidly prototype and diversify gene expression at the pathway level and an in vivo genome reshuffling system to integrate assembled pathways into the synthetic yeast genome while combinatorially causing massive genome rearrangements in the host chassis. A set of loxP mutant pairs was identified to maximize the efficiency of the in vitro diversification. Exemplar pathways of β-carotene and violacein were successfully assembled, diversified, and integrated using this SCRaMbLE-in method. High-throughput sequencing was performed on selected engineered strains to reveal the resulting genotype-to-phenotype relationships. The SCRaMbLE-in method proves to be a rapid, efficient, and universal method to fast track the cycle of engineering biology.

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Whole genome synthesis: from poliovirus to synthetic yeast

Cited by 3 publications

References 49 publications

Improving Chromosome Synthesis with a Semiquantitative Phenotypic Assay and Refined Assembly Strategy

Improving Chromosome Synthesis with a Semiquantitative Phenotypic Assay and Refined Assembly Strategy

Whole genome engineering by synthesis

Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods

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