Abstract
SCRaMbLE is a novel system implemented in the synthetic yeast genome, enabling massive chromosome rearrangements to produce strains with a large genotypic diversity upon induction. Here we describe a reporter of SCRaMbLEd cells using efficient selection, termed ReSCuES, based on a loxP-mediated switch of two auxotrophic markers. We show that all randomly isolated clones contained rearrangements within the synthetic chromosome, demonstrating high efficiency of selection. Using ReSCuES, we illustrate the ability of SCRaMbLE to generate strains with increased tolerance to several stress factors, such as ethanol, heat and acetic acid. Furthermore, by analyzing the tolerant strains, we are able to identify ACE2, a transcription factor required for septum destruction after cytokinesis, as a negative regulator of ethanol tolerance. Collectively, this work not only establishes a generic platform to rapidly identify strains of interest by SCRaMbLE, but also provides methods to dissect the underlying mechanisms of resistance.
Highlights
SCRaMbLE is a novel system implemented in the synthetic yeast genome, enabling massive chromosome rearrangements to produce strains with a large genotypic diversity upon induction
Activating ReSCuES by Cre-mediated recombination between the two loxP sites will result in the inversion of the “URA3-LEU2” module, which will subsequently turn on the expression of LEU2 gene while, simultaneously, shutting down URA3 expression
Only strains with activated ReSCuES can survive in media lacking leucine (Fig. 1, SCRaMbLEd yeast)
Summary
SCRaMbLE is a novel system implemented in the synthetic yeast genome, enabling massive chromosome rearrangements to produce strains with a large genotypic diversity upon induction. We show that all randomly isolated clones contained rearrangements within the synthetic chromosome, demonstrating high efficiency of selection. Previous work demonstrated the ability of SCRaMbLE to generate phenotypic diversity, producing strains with heterogeneous growth rates[19], and to delete unessential genomic regions flanked by specific loxPsym sites[20,23]. There are SCRaMbLE “escapers”, which survived due to the mutated Cre gene These un-scrambled cells can interfere with the identification of strains with rearranged genomes. ReSCuES has been developed to efficiently identify SCRaMbLEd colonies in strains containing synXII, the largest synthetic chromosome in Saccharomyces cerevisiae. ReSCuES is applied to isolate yeast strains with increased tolerance to several stress
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