Team:Washington/Our Project

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<h1> <center>Our System </center></h1>
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<h2>Natural Cell Processes</h2>
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We sought to make use of a cell's natural methods of degrading misfolded proteins. If a protein is misfolded in a cell it is targeted by the E3 ligase which attaches a ubiquitin to it. This marks the protein for degradation by the proteasome. If a protein is unstable it is likely at very low levels within the cell as it is being degraded by this system.
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<h2>Gal4-VP16</h2>
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Our system relies on GFP being produced at different levels depending on the stability of a protein of interest. To do this our system puts the protein of interest in between Gal4 and VP16.<br> </p>
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  <center> <img src="https://static.igem.org/mediawiki/2014/d/d2/Frontcover.jpg" width="50%" alt="Gal4-VP16 Construct">
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  <sup> <b> Fig 1. Our project utilizes the Gal4-VP16 transcriptional activator to test protein stability in terms of GFP output. </b> </sup> </center>
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<p>Together Gal4 and VP16 up-regulate the expression of a gene under certain promoters, in our case we used Gal1. Gal4 binds to the DNA and then VP16 recruits RNA polymerase to begin transcription of the gene under Gal1. Independently Gal4 simply binds to Gal1, and VP16 is not localized to the DNA. So if our gene located in between the two, degradation of the gene leads to no transcription of anything under Gal1.
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</p>
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  <h1> Background </h1>
 
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<p align = left> New novel methods must be first tested for viability against other existing methods. Our project is no different. In order, to gauge the effectiveness and accuracy of our method we
 
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choose test proteins that are well studied and characterized. Therefore, BINDI and several of its mutant variants that have been well studied were chosen. The first step of our
 
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project was to replicate the results of the studies on BINDI and its variants by repeating the stability test experiments presented in "the paper." After verifying the results
 
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of "the paper", we proceeded to construct our degron protein constructs and expressed them in yeast cells containing an inducible mechanism for the expression of green fluorescence
 
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protein. Subsequently, the fluorescent emission of each cell is measured as higher fluorescent corresponds to higher test protein stability. </p>
 
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  <h2> Our System </h2>
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<h2>Using a Degron to Exaggerate Differences in Stability</h2>
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<h3> Components of the Degron Construct </h3>
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<p>
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<h3> Test Protein </h3>
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We sought to make a versatile system that could be used for proteins of various native stabilities. If a protein is stable enough to avoid ubiquitination but not stable enough for its engineered purpose our system would not be useful. To deal with this we used a degron. <br>
 +
<br>
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A degron is an inherently unstable protein domain. By inserting this into the fusion protein produced by our plasmid we expect a protein which is just stable enough to avoid degredation will become unstable and be degraded. We expect a very stable protein to be able to overcome this source of instability and this will allow us to the measure differences in stability of more stable protein variants where without the degron they would give very similar measurements in our system.
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<h2> Components of Our Plasmid </h2>
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              <p align = left> 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 established protein stability testing methods. Therefore,our team decided to use the protein known as BINDI. </p>
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The fusion protein produced by expression of our plasmid is made up of the Gal4-VP16 transactivating complex with a protein of interest in between. Positioning of the degron is determined by the native stability of the protein of interest.  
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  <h2> Method </h2>
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  <p align =left> The essential process of our system involves cloning and manufacturing of a plasmid in <i> E. coli </i>. Once, the plasmids have been constructed and verified in <i> E. coli </i>
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</p>
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they are transformed into <i>S. cerevisiae</i>. The plasmid constructs are then expressed. Following several days of growth the yeast cultures are passed through a Flow Cytometer
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  <center> <img src="https://static.igem.org/mediawiki/2014/1/1d/UWPlasmid.png" alt="Degron Constructs" style="width:40%">
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and the fluorescence of each cell is measured. Higher fluorescence is associated with higher expression of the protein of interest which in-turn is indicative of higher protein
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stability. </p>
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  <br>
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<h3> Cloning in <i> Escherichia coli </i> </h3>
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<p align = left> There are five possible degron constructs corresponding to five different positions the degron can take in our construct. The first step is to insert our protein into each
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of the five constructs and verify it. </p>
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<h3> Preparation and Passaging of <i> Saccharomyces cerevisiae </i> </h3>
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<p align = left> Once, plasmids of the five possible degron constructs have been cloned with our three test proteins, they are subsequently transformed in PyE1 a strain of
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<i> S. cerevisiae </i> with the ability to produce green fluorescent proteins with the proper promoter protein such as Gal4 which is a part of our degron construct.
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Following the transformation, the cells are plated onto plates with on a  Selective Dropout C-Uracil media and incubated at 30<sup>o</sup>C for 2 days. After two days, three
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colonies are chosen and added to an overnight culture of 3mL Selective Dropout Media C-Uracil and 2% Glucose then incubated for another two days at 30<sup>o</sup>C.
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After two days of incubation, a 20-50uL aliquot of each culture is "passaged" into another 3mL culture prepared in the same manner as before and incubated for the same
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duration and temperature as the previous culture. The passaging is done several times after each passage after the second passage, a glycerol stock is prepare from the
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culture and Flow Cytometry is run on the culture.</p>
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<p align = left> The purpose of passaging is to gradually remove excess copies of the plasmid constructs. Excess copies, exceeding one per cell will lead to multiple fold increase in the
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expression of the degron protein construct. As a result of this, GFP expression will also be increased thus reducing the viability and accuracy of the Flow Cytometry
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measurements conducted on each cell culture.</p> 
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      <h3> Relative Stability Analyzed via Flow Cytometry </h3>
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  <sup> <b> Fig 2. Potential Degron insert sites for our system. </b> </sup> </center>
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  <br> <br>
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      <h3> Mutagenesis through Error Prone Polymerase Chain Reactions </h3>
 
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      <h3> Selecting Stable Variants through Fluorescence Activated Cell Sorting </h3>
 
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  <h2> Results </h2>
 
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<h3> Protein stability analysis using Circular Dichroism </h3>
 
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              <p align = left> Our system was verified using Circular Dichroism (CD) analysis. A scan of the protein in solution in PBS was scanned across a variety of wavelengths to find the signal minima that would best indicate the state of folding. An equivalent concentration of protein in concentrated guanidinium chloride (GdmCL), a powerful chaotropic agent, was then prepared to be mixed in to our sample. This solution was then added to the sample in small increments, allowing us to measure the CD signal at increasing concentrations of GdmCl while maintaining a constant concentration of the protein being tested. The concentration of GdmCl at which the CD signal was half of its initial value was recorded. A higher concentration of GdmCl being required to half denature a protein indicates greater stability.</p>
 
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        <h3> Protein expression analysis using SDS-PAGE </h3>
 
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<p align = left>
 
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Samples of our protein of interest were taken at various points during the purification cycle. These samples were then run through gel electrophoresis to determine the relative amounts of protein produced by the cells. Larger bands were indicative of greater expression.
 
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</p>
 
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<h3> Protein stability analysis using Degron Constructs and Flow Cytometry </h3>
 
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<h3> Mutagenesis Results and Mutant Variant Analysis</h3>
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<p>
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  <h2> Future Plans </h2>
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  There are 5 possible degron positions: <br>
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<h3> Examination of more proteins </h3>
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  -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). <br>
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  -Deg1: This construct contains the Degron in front of our Gal4-Protein-VP16 complex. <br>
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  -Deg2: This construct contains the Degron in between Gal4 and the protein in our Gal4-Protein-VP15 complex. <br>
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  -Deg3: This construct contains the Degron in between the protein and VP16 in our Gal4-Protein-VP15 complex. <br>
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  -Deg4: This construct contains the Degron at the end of our Gal4-Protein-VP16 complex. <br>
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</p>
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  <h3> Relative Stability Analyzed via Flow Cytometry </h3>
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<center><img src="https://static.igem.org/mediawiki/2014/d/d9/Degron_construct.jpg" alt="Degron Constructs" style="width:50%">
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<br>
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<sup> <b> Fig 3. Expected GFP output based on our Degron constructs </b></sup> </center>
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<br>
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                <p>
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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. <br>
 +
<br>
 +
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, therefore we expect it to be 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.<br>
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      </p>
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<h3> Further evolving more stable variants of existing proteins </h3>
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  <h2> Submitted Parts </h2>
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<h3> Degron (BBa_K1408000) </h3>
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<h2> Test Protein </h2>
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<p> The Degron is an unstable protein domain that, when fused to a protein, acts as a source of instability. The presence of the Degron leads to degradation of the protein by the cell via ubiquitination. This is a new part we are submitting to the registry. </p>
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<h3> Gal4-VP16 Transcriptional Activator (BBa_K1408001) </h3>
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<p align = left>  
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<p> This part consists of our Gal4-VP16 fusion protein, which is a translational activator. Gal4 protein binds to a Gal1 promoter, while VP16 recruits transcription machinery, promoting transcription to anything under the Gal1 promoter. The two must be co-localized to work as a transcriptional activator. </p>
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<h3> Gal4-Degron-VP16 (BBa_K1408002) </h3>
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  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.
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<p> This part is a combination of our previous two parts submitted to the registry. By utilizing this system, the relative stabilities of proteins can be compared. A sequence for a protein inserted between the Degron and VP16 will produce a fusion protein which acts as a transcriptional activator for anything under a Gal1 promoter. If the protein inserted is unstable it will be degraded by the cell and the Gal4-VP16 transcriptional activator will no longer function. If a quantifiable marker protein such as Green Fluorescent Protein (GFP) is under Gal1 then the level of GFP output of the cell is related to the stability of the inserted protein. </p>
+
  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 al<sup>1</sup>.
 +
  We would like to acknowledge and thank Dr. Procko for giving us his genes to work with.
 +
 +
</p>
 +
 
 +
 
 +
        <h2> PyE1 a strain of <i> Saccharomyces cerevisiae </i> </h2>
 +
<p>
 +
 
 +
  We use a strain of <i> Saccharomyces cerevisiae </i> deveoloped in Stan Fields' lab at the University of Washington called PyE1.  
 +
                  Its genome has been engineered to contain a gene from Green Fluorescent Protein (GFP) under a Gal1 promoter.  
 +
                  When the Gal4 DNA-binding domain and the VP16 transcription activation domain are colocalized to the Gal1 promoter, expression of GFP is induced.
 +
  Therefore, using our test plasmids in PyE1 generates GFP relative to the level of Gal4/VP16 peptide in 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.
 +
 +
</p>
 +
 
 +
        <p>
 +
        <sup>1</sup>Procko, 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.
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Latest revision as of 02:41, 18 October 2014

UW Homepage Official iGEM website

Our System

Natural Cell Processes

We sought to make use of a cell's natural methods of degrading misfolded proteins. If a protein is misfolded in a cell it is targeted by the E3 ligase which attaches a ubiquitin to it. This marks the protein for degradation by the proteasome. If a protein is unstable it is likely at very low levels within the cell as it is being degraded by this system.

Gal4-VP16

Our system relies on GFP being produced at different levels depending on the stability of a protein of interest. To do this our system puts the protein of interest in between Gal4 and VP16.

Gal4-VP16 Construct
Fig 1. Our project utilizes the Gal4-VP16 transcriptional activator to test protein stability in terms of GFP output.


Together Gal4 and VP16 up-regulate the expression of a gene under certain promoters, in our case we used Gal1. Gal4 binds to the DNA and then VP16 recruits RNA polymerase to begin transcription of the gene under Gal1. Independently Gal4 simply binds to Gal1, and VP16 is not localized to the DNA. So if our gene located in between the two, degradation of the gene leads to no transcription of anything under Gal1.

Using a Degron to Exaggerate Differences in Stability

We sought to make a versatile system that could be used for proteins of various native stabilities. If a protein is stable enough to avoid ubiquitination but not stable enough for its engineered purpose our system would not be useful. To deal with this we used a degron.

A degron is an inherently unstable protein domain. By inserting this into the fusion protein produced by our plasmid we expect a protein which is just stable enough to avoid degredation will become unstable and be degraded. We expect a very stable protein to be able to overcome this source of instability and this will allow us to the measure differences in stability of more stable protein variants where without the degron they would give very similar measurements in our system.

Components of Our Plasmid

The fusion protein produced by expression of our plasmid is made up of the Gal4-VP16 transactivating complex with a protein of interest in between. Positioning of the degron is determined by the native stability of the protein of interest.

Degron Constructs
Fig 2. Potential Degron insert sites for our system.


There are 5 possible degron positions:
-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

Degron Constructs
Fig 3. 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, therefore we expect it to be 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

We use a strain of Saccharomyces cerevisiae deveoloped in Stan Fields' lab at the University of Washington called PyE1. Its genome has been engineered to contain a gene from Green Fluorescent Protein (GFP) under a Gal1 promoter. When the Gal4 DNA-binding domain and the VP16 transcription activation domain are colocalized to the Gal1 promoter, expression of GFP is induced. Therefore, using our test plasmids in PyE1 generates GFP relative to the level of Gal4/VP16 peptide in 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.

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