OFF to PriON

Waterloo iGEM 2016: Using stop codon read-through and CRISPR to explore S. cerevisiae prion mechanisms

Motivated by human neurodegenerative diseases (Alzheimer's, Creutzfeldt-Jakob, and Parkinson's) induced by prionic protein states in the brain, the Waterloo iGEM Team designed a negative feedback control using novel regulatory elements designed to over-express or repress target proteins during the transition from healthy state to prionic state. Using the Sup35 prion protein associated with [PSI-] (healthy) and [PSI+] (diseased) states of S. cerevisiae cells, Waterloo iGEM used read-through of a premature stop codon combined with Hsp104 and dCas9 respectively to overexpress or repress Hsp104. Overexpression and repression of Hsp104 both cure the [PSI+] state from priON to OFF.

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 read-through. 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.

Involvement: Waterloo iGEM Lab and Design team, conducting work-ups, nanodrop measurements, and supporting lab work.

Waterloo iGEM information and resources:

• Website: http://igem.uwaterloo.ca/

• Archival Waterloo iGEM/Wiki: http://2016.igem.org/Team:Waterloo

• GitHub Repository: https://github.com/igem-waterloo/uwaterloo-igem-2016/tree/master/Wiki

• Biobrick contributions from iGEM Waterloo Team:

  • Part:BBa_K2137001 CUP1-GFP Transcriptional Fusion parts.igem.org/Part:BBa_K2137001

  • Part:BBa_K2137002 Gal1-GFP Transcriptional Fusion parts.igem.org/Part:BBa_K2137002