Team:Waterloo
OFF to priON
Using stop codon read-through and CRISPR to explore S. cerevisiae prion mechanisms
Furthering the advancement of research into prions and neurodegenerative diseases
We initiate a [PSI+] response in S. cerevisiae using a plasmid that will overexpress Sup35-NM.
We insert a premature stop codon into an N-terminal CFP tag that will be read through and fluoresce during a [PSI+] response
We control read through rates by choosing the location of the premature stop codon, and can therefore control protein production in [PSI+]. We use Hsp104, a chaperone protein in S. cerevisiae, to demonstrate that our set-up phenotypically responds to the stop codon readthrough.
ABSTRACT
Prions, or “zombie proteins,” are infectious agents that lead to a variety of neurodegenerative disorders (NDDs). They sicken cells by aggregating with each other and prevent proper protein folding leading to cell death from the accumulated damage. We propose a synthetic biology approach to better study prion propagation in the model organism S. cerevisiae. Our system involves inserting a premature stop codon into a protein open reading frame of interest or into dCas9 to respectively overexpress or knock-down protein levels during a [PSI+] response. We use Hsp104, a chaperone protein in S. cerevisiae, to demonstrate that our set-up phenotypically responds to the stop codon readthrough. This research is useful for continuously observing a phenotypic output during prion propagation in yeast and may have implications for helping to identify protein targets for both prevention and treatment of NDDs in the future.
