Workflow 3: Programmed genome rearrangement in yeast

Yeast Scramble Lab Manual

Mark Jones, Lisa Scheifele

Version: 1.0

This laboratory manual includes protocols for SCRaMbling of yeast cells as well as phenotypic characterization. Appropriate student exercises are also included. This resource was developed as part of NSF Award #1827204, RCN-UBE: The Build-a-Genome Network
introductory, Lab, Teaching material, Undergraduate, Advanced, Majors, Extended Project
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07.2020

There are many unanswered questions about the arrangement and structure of complex genomes. One way to investigate these issues is to manipulate the arrangement and order of genes and functional elements within a genome. The SCRaMbLE system (”Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution”) allows us to do this.

 
Luo et al., Nature Communications, 2017, DOI:10.1038/s41467-017-00806-y 
Article licensed under a Creative Commons Attribution 4.0 International License.
 

The Synthetic Yeast Project has introduced LoxP sites throughout the redesigned synthetic yeast genome. We have been working with yeast strains containing synthetic versions of chromosomes V and X which contain hundreds of LoxP sites. Becuase the Cre gene is under the control of an inducible promoter, rearrangements between the LoxP sites can be induced, leading to a population of yeast with diverse chromosomal structures and arrangements. When we plate cells after SCRaMbLE, we typically see heterogeneous colony sizes, indicative of changes in fitness under our selective conditions; some strains grow more poorly, but some strains have enhanced growth in the selective conditions.

                                     

Blount et. al., Nature Communications 9, Article number: 1932 (2018) 
Article licensed under a Creative Commons Attribution 4.0 International License.

 

 

This yeast population can then be screened for particular phenotypes under selective conditions. We typically confirm that our selected yeast have enhanced growth under selective conditions by performing growth assays, such as our plating of serially diluted yeast strains below. Students can then investigate what genomic changes underlie these new properties.