Exploring the genome rearrangement system, SCRaMbLE, to introduce DNA into Saccharomyces cerevisiae
thesisposted on 2022-03-28, 11:36 authored by Elizabeth Louise Izolde Wightman
A novel genome rearrangement tool, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP mediated evolution), has been designed for the synthetic genome of S. cerevisiae. Collaborators of the 'yeast 2.0' project initially construct one version of the synthetic genome. However, SCRaMbLE can rapidly generate billions of unique genomes in even a small culture. Only one of the 16 chromosomes is currently complete, limiting full genome rearrangement by SCRaMbLE. As a contribution to the international project, I constructed and integrated a 'megachunk' of synthetic chromosome 14 into S. cerevisiae. Currently, SCRaMbLE is limited to S. cerevisiae DNA only. I aimed to explore SCRaMbLE in a novel application: as a tool to introduce foreign DNA into S. cervisiae. To develop protocols, the S. cerevisiae marker gene URA3 was used to SCRaMbLE into yeast. SCRaMbLEing URA3 increasing the transformation efficiency significantly compared to non-SCRaMbLEd URA3.gDNA from the filamentous fungus Trichoderma reesei was used as an example of SCRaMbLEing large libraries of foreign DNA into yeast. Successful attachment of SCRaMbLE recombination sites to gDNA fragments was achieved using a cloning approach, gDNA fragments were SCRaMbLEd into yeast and cellulase activity was screened.