Part:BBa_K2475000
Sup 35 Prion Domain (PrD)
The Sup35 prion domain (PrD) contains the first 137 amino acids of the Sup35 protein. This domain gives the Sup35 protein the ability to aggregate. By fusing it to other proteins this aggregation ability should be transferred to other proteins. PrD fused to other proteins should have minimal impact of the folding structure, however loss of function may occur in the prion state due to localization of the protein in an aggregate composed of proteins with this domain attached.
To verify whether or not the fluorescent proteins we attached to Sup35’s prion domain still fluoresce, we expressed them under the Cup1 promoter in the W303 strain of yeast. We had variants of the strain that were both [PSI+] and [psi-] (ie. strains that had aggregated and soluble Sup35 respectively), and expressed our constructs in both.
As stated in our section on Cup1 promoter characterization, we observed that empty yeast cells never had more than 0.2% of their population expressing a fluorescence greater than 1000 fluorescence units as measured by our flow cytometer. Therefore, we defined “fluorescent” cells as cells having a fluorescence of greater than 1000 fluorescence units.
We observed yeast samples consisting of 10,000 cells in a flow cytometer that used a 488 nm laser. This laser is capable of moderately inefficient excitation of YFP and very inefficient excitation of CFP. Monitoring of fluorescence was performed with a channel that accepted any emission from 505 nm to 560 nm.
The data we collected are formatted as follows. Y is used as a shorthand for our construct [PrD-YFP] (https://parts.igem.org/Part:BBa_K2475002), and C is used as a shorthand for our construct [PrD-CFP](https://parts.igem.org/Part:BBa_K2475001). The samples are identified by which constructs they contain. There were two separately performed transformations of PrD-YFP into [psi-] yeast to verify that that particular part is fully functional. [psi-] YFP R (“R” standing for replicate here) was done separately, and all other samples were transformed together on the same day. Average fluorescence is provided in fluorescence units as measured by the flow cytometer. Additionally, the average fluorescence provided is the average of the fluorescent population **ONLY**.
The presence of our constructs in our yeast was confirmed by nutritional selection during transformation, and replica plating of samples after transformation onto nutritionally selective media. All transformations were efficient (100s to 1000s of colonies), indicating no substantial problem.
Samples were taken directly from transformation plates and suspended in phosphate buffered solution (PBS), as this was the buffer used in the flow cytometer. The reason why samples were taken straight from the plate was because fluorescence decreased rapidly once samples were inoculated in YPD (though it persisted at a low level in most samples after 12 hours).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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