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Revision as of 05:56, 17 October 2014

Our System

Components of the Degron Construct

^{Fig 1. Potential Degron insert sites for our system.}

Our experiment utilizes 5 different Degron constructs:
-Deg0: This construct contains only the Gal4-VP16 transcriptional activator complex with the protein of interest in between the two (shortened as Gal4-Protein-VP16).
-Deg1: This construct contains the Degron in front of our Gal4-Protein-VP16 complex.
-Deg2: This construct contains the Degron in between Gal4 and the protein in our Gal4-Protein-VP15 complex.
-Deg3: This construct contains the Degron in between the protein and VP16 in our Gal4-Protein-VP15 complex.
-Deg4: This construct contains the Degron at the end of our Gal4-Protein-VP16 complex.

Relative Stability Analyzed via Flow Cytometry

^{Fig 2. Expected GFP output based on our Degron constructs}

Flow cytometry is a high throughput method of analyzing cells for various optical outputs, namely fluorescence. A flow cytometer is an analytical instrument in which cells that have been suspended in a solution are passed through a narrow channel in which fluorescence of individual cells can be measured.

By utilizing Flow Cytometry, we can measure the amount of GFP output within cells from each degron construct. Based on where the Degron is inserted, we expected a different level of fluorescence. As such, we expected to see the highest GFP production in our Deg0 construct, as it only contains the Gal4-Protein-VP16 complex with no Degron inserted, hence being the most stable. We expected that Deg2 and Deg3 would have a lower GFP production than Deg0 but higher than Deg1 and Deg4. This rationale was based on the fact that the Deg1 and Deg4 have the Degron exposed, making it more likely to be degraded by ubiquitination than in Deg2 and Deg3 which has the Degron buried inside the Gal4-Protein-VP16 complex.

Test Protein

The test protein that must be chosen in testing a novel and new system must be a protein that has been well studied and rigorously examined through other existing and well accepted protein stability testing methods. Therefore,our team decided to use the protein known as BINDI. BINDI and two of its less stable variants, BbpD04 and BbpD04.3 were studied and examined in "A Computationally Designed Inhibitor of an Epstein-Barr Viral Bcl-2 Protein Induces Apoptosis in Infected Cells" by Procko et al¹. We would like to acknowledge and thank Dr. Procko for giving us his genes to work with.

PYE1 a strain of Saccharomyces cerevisiae

The lynchpin of our project is the usage of flow cytometry and fluorescence activated cell sorting for high throughput protein stability analysis. However, both analytical systems, flow cytometry and F.A.C.S. require fluorescence emission. Therefore we need a method of generating fluorescence within our cells in a way that also gives us insight into protein stability. Each degron construct contains a Gal4 promoter which can bind to an upstream activating site, Gal1, that induces downstream expression of something. It just so happens that PYE1, this something happens to be Green Fluorescent Proteins. Protein expressions in PYE1, will allow us to generate GFP relative to the amount of degron protein construct that exist within the cell. The more stable the degron protein construct is, the more likely it is that more GFP will be expressed. This relationship between stability and GFP forms the basis from which we will measure the relative protein stability of our degron constructs as well as the protein of interest degron construct.

¹Procko, E, et al. "A Computationally Designed Inhibitor of an Epstein-Barr Viral Bcl-2 Protein Induces Apoptosis in Infected Cells" Cell 157 (2014): 1644-56.