Abstract
SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution) is a genome restructuring technique that can be used in synthetic genomes such as that of Sc2.0, the synthetic yeast genome, which contains hundreds to thousands of strategically positioned loxPsym sites. SCRaMbLE has been used to induce rearrangements in yeast strains harboring one or more synthetic chromosomes, as well as plasmid DNA in vitro and in vivo. Here we describe a collection of heterozygous diploid strains produced by mating haploid semisynthetic Sc2.0 strains to haploid native parental strains. We subsequently demonstrate that such heterozygous diploid strains are more robust to the effects of SCRaMbLE than haploid semisynthetic strains, rapidly improve rationally selected phenotypes in SCRaMbLEd heterozygous diploids, and establish that multiple sets of independent genomic rearrangements are able to lead to similar phenotype enhancements. Finally, we show that heterozygous diploid SCRaMbLE can also be carried out in interspecies hybrid strains.
Highlights
SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution) is a genome restructuring technique that can be used in synthetic genomes such as that of Sc2.0, the synthetic yeast genome, which contains hundreds to thousands of strategically positioned loxPsym sites
Controlling the activity of Cre is important for maintaining Sc2.0 chromosome stability; to implement this, Cre is fused to the estrogen binding domain (EBD)[5] of the estrogen receptor, which effectively sequesters CreEBD in the cytosol
Characterization of SCRaMbLE in diverse heterozygous yeast. Each member of this collection was produced by mating a haploid strain bearing either one or two synthetic chromosomes[8,9] with a haploid strain from the Saccharomyces Genome Resequencing Project (SGRP) set[10,11]
Summary
SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution) is a genome restructuring technique that can be used in synthetic genomes such as that of Sc2.0, the synthetic yeast genome, which contains hundreds to thousands of strategically positioned loxPsym sites. SCRaMbLE of haploid strains bearing one or more synthetic chromosomes results in a high lethality rate due to the deletion of one or more essential genes[3,6]. We demonstrate that SCRaMbLE in heterozygous diploids results in a higher proportion of surviving cells in strains bearing both one and two synthetic chromosomes.
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