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From 2014.igem.org

Our System

Components of the Degron Construct

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.

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 "CITE THE BINDI PAPER." Many of the individuals who were cited in the paper as well as the lab that did the research was done in was a nearby lab at the University of Washington. Therefore, it was convient for us to contact members of the Baker Lab and speak with them about the protein we intended on using as well as accquiring samples of the protein's DNA code.

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 emmission. 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.