Team:Northern BC/Design
Design
Our opioid biosensor design is relatively simple: hijack the intracellular machinery of yeast cells early in an endogenous signaling pathway in order to amplify the opioid-binding signal as much as possible. Our pathway has been engineered to ultimately end with a visual change in color as chromoprotein production is turned on if opioids are present in a drug sample. To achieve this, our design has three discrete components that must come together in the working prototype:
1. We needed to disrupt the endogenous signaling pathway such that the natural ligand no longer activates the system in an effort to eliminate false positives. To achieve this, we chose a signaling pathway that is not essential for yeast survival and will not affect normal yeast functions. We targeted the receptor of this endogenous pathway for disruption by homologous recombination in which we intended to insert the KanMX6 gene within the open reading frame of the target, thereby turning off its production. This would allow us to express our opioid detector in yeast such that an opioid/detector binding event is the only trigger to activate the signaling pathway.
2. We needed to clone our opioid detector into a yeast shuttle vector for constitutive expression in yeast cells. Since the shuttle vector we intended to use for detector expression was not RFC-10-compatable, we had to remove two restriction sites in the backbone before inserting our gene (See our Results page).
3. We needed to clone our reporter (aeblue chromoprotein) into a yeast shuttle vector for expression in yeast cells. We placed this gene under the control of a modified CYC1 promoter that contains a binding site for a yeast transcription factor. In this way, chromoprotein production is only initiated when the transcription factor is activated, which only occurs if opioids are present in the system. Since the shuttle vector we intended to use for reporter expression was not RFC-10-compatable, we had to remove two restriction sites in this backbone as well before inserting our gene (See our Results page).
