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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<center><img src="https://static.igem.org/mediawiki/2014/d/d9/Degron_construct.jpg" alt="Degron Constructs" style="width:500px;height:290px"></center>
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<p> Our experiment utilizes 5 different Degron constructs: <br>
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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>
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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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<br>
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-Deg1: This construct contains the Degron in front of our Gal4-Protein-VP16 complex. <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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-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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<h2> Components of Our Plasmid </h2>
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<p>
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<h3> Test Protein </h3>
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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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              <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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</p>
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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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        <h3> PYE1 a strain of <i> Saccharomyces cerevisiae </i> </h3>
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<p>
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The lynchpin of our project is the usage of flow cytometry and fluorescence activated cell sorting for high throughput analysis.  
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However, both analytical systems 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.
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Each degron construct contains a Gal4 promoter which binds to an upstream activating site that induces downstream expression of something.
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If just so happens that in PYE1, this something happens to be Green Fluorescent Proteins.
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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 the more likely that more GFP will be expressed.
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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.
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</p>
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      <h2> Method </h2>
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  <br>
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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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  <sup> <b> Fig 2. Potential Degron insert sites for our system. </b> </sup> </center>
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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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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> <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 of the five constructs and verify that our protein of interest is in the correct location in the correct construct. Once, verified a miniprep of each protein of interest in each degron construct is cultivated from an <i> E.Coli </i> culture. </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, GFP expression will also be increased without reflecting a change in the protein's stability thus reducing the viability and accuracy of the Flow Cytometry
 
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measurements conducted on each cell culture containing our protein of interest-degron construct.</p> 
 
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      <h3> Relative Stability Analyzed via Flow Cytometry </h3>
 
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<p> Flow cytometry is a high throughput method of analyzing cells for various optical outputs, namely fluorescnce. 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 invidual cells can be measured. <br>
 
-
 
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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 Deg1 and Deg4 would have a lower GFP production than Deg0 but higher than Deg2 and Deg3. This rationale was based on the fact that the Degron would be more destabilizing for the Gal4-Protein-VP16 complex if the Degron is found in between this complex rather than outside of it. <br> </p>
 
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        <h3> Mutagenesis through Error Prone Polymerase Chain Reactions (PCRs) </h3>
 
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<p>
 
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In order to validate our system as being capable of selecting more stable protein variants, we have to produce mutations in our protein of interest.
 
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Those mutations could potentially be beneficial and could carry a stabilizing affect on the protein of interest.
 
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Error-prone PCR utilizes DNA-polymerase's error-prone nature and further increases the likelyhood of mutations by manipulating the conditions in which,
 
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DNA-polymerase operates in, thereby causing the polymerase to create errors in DNA sequencing which in turn will create changes in the protein construct.
 
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Once, the DNA coding of our protein has been changed we can express the mutations and analyze those stabilizing or de-stabilizing affects they have on our protein.
 
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The possible mutations can then be analyzed using the degron system and more stable mutations can be easily seen then sequenced.
 
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</p>
 
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<p>
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      <h3> Selecting Stable Variants through Fluorescence Activated Cell Sorting </h3>
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<p>
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  There are 5 possible degron positions: <br>
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Once, the DNA coding for a protein of interest has been mutated and the protein expressed within PYE1, we can then analyze the fluorescent output and select cells
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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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that exhibit higher fluorescence. The nature of our system allows us to conclude that higher fluorescence output corresponds to higher stabily
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  -Deg1: This construct contains the Degron in front of our Gal4-Protein-VP16 complex. <br>
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of the protein of interest. Cells with higher fluorescence are then selected, cultured and the plasmid within them is sequenced.
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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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Fluorescence Activated Cell Sorting (F.A.C.S.) makes our system very high thoughput and allows us to analyze a large number of cells and possible mutations.  
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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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</p>
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  -Deg4: This construct contains the Degron at the end of our Gal4-Protein-VP16 complex. <br>
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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>
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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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<p>
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  <h3> Relative Stability Analyzed via Flow Cytometry </h3>
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The results for the no-protein of interest degron constructs matched expected results in which Deg0 had the highest expect stability which correlated to the highest
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fluorescence output. These expectations were validated by our experiment results in which cells with Deg0 exhibited the highest fluorescent levels.
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Our second expectations were that Deg2 and Deg3 would exhibit middle levels of fluorscence, lower than Deg0 but higher than Deg1 or deg4. Once again, these
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expectations were validated by our experimental results.
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The results for the no protein of interest degron constructs are as follow, cells containing our Deg0 protein construct exhibited the highest fluorescent
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followed by Deg2 and deg3 and finally by Deg1 and Deg4 both of which exhibited baseline levels (no protein, no degron construct PYE1 cells) of fluorescence.
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<br>
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In order, to validate the system as whole we must analyze the degron constructs with a specific well studied protein to analyze that protein's stability with our
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system compared to the protein stability measurements from current existing techniques.
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There were three variants of a protein each with varying stabilities that were quantified using circular dichroism and guanidium hydrogen chloride melts.
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The protein BINDI had the highest stability followed by BbpD04.3 and then BbpD04. If our system is accurate, cells containing the BINID-Degron construct would
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exhibit the highest fluorescence output followed by BbpD04.3 and BbpD04 would show the lowest fluorescence output.
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Since there are 5 possible degron construct for each of our 3 proteins of interest, all 15 data points would have to match our expectations.
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We expect that BINDI Deg0 would have the highest fluorescence of the all protein of interest Deg0 constructs followed by BbpD04.3 Deg0 and BbpD04 Deg0.
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Next, BINDI Deg2/3 would have middle levels of fluorescence followed by BbpD04.3 Deg2/3 and BbpD04 Deg2/3.
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Finally, BINDI Deg1/4, BbpD04.3 Deg1/4 and BbpD04 Deg1/4 would have the lowest levels of fluorescence if not baseline levels.
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The experimental results accquired through flow cytometry show a rough correlation to these expections********more experiment required***********
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</p>
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<h3> Mutagenesis Results and Mutant Variant Analysis</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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  <h2> Future Plans </h2>
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<h3> Examination of more proteins </h3>
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                      <p>
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<br>
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Like any new up and coming technique, the degron system, will require further testing with a larger variety of well studied mutant variants of a single protein as well as a larger number of well studied proteins in general before the system can truly be accepted. <br>
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<sup> <b> Fig 3. Expected GFP output based on our Degron constructs </b></sup> </center>
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Our current plans for the future are too test a protein 33RM2 and its less stable variant 33CL1 both of which are bind to PD-1 (a negative t-cell regulator that prevents the recognition of tumorous cells by the immune system).
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Since, the stability of both these proteins are known and have been verified using other techniques such as thermal melts, they are very suitable candidates for testing using our degron system. <br>
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<br>
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</p>
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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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                        <p>
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Throughout this past summer, out team has been evolving more stable variants of BINDI through error-prone PCR and going forwards we will continue this process and continually analyze the mutants with flow cytometry and select cells that exhibit higher fluorescence with FACS.
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Furthermore, our technique could be applied...<br>
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***********NEEDS TO BE FINISHED***************
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</p>
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  <h2> Submitted Parts </h2>
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<center><img src="https://static.igem.org/mediawiki/2014/a/a2/Bio-brick_image.jpg" alt="Submitted Biobricks" style="width:500px;height:226px"></center>
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<h2> Test Protein </h2>
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<h3> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1408000" target="_blank">Degron (BBa_K1408000)</a> </h3>
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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> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1408001" target="_blank"> Gal4-VP16 Transcriptional Activator (BBa_K1408001) </a> </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 transcriptional 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. <br>
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This transcrptional activator has been previously submitted to the registry (BBa_K1179014 submitted by the iGEM 2013 MIT team). However, we noticed that this part was incomplete compared to ours and that this part had no user data related to it. In order to make the complete part available, together with experimental data related to the part, we decided to "re-submit" this part to the registry. </p>
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<h3> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1408002" target="_blank"> Gal4-Degron-VP16 (BBa_K1408002) </a> </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>
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        <h2> PyE1 a strain of <i> Saccharomyces cerevisiae </i> </h2>
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<p>
 +
 
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  We use a strain of <i> Saccharomyces cerevisiae </i> deveoloped in Stan Fields' lab at the University of Washington called PyE1.
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                  Its genome has been engineered to contain a gene from Green Fluorescent Protein (GFP) under a Gal1 promoter.
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                  When the Gal4 DNA-binding domain and the VP16 transcription activation domain are colocalized to the Gal1 promoter, expression of GFP is induced.
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  Therefore, using our test plasmids in PyE1 generates GFP relative to the level of Gal4/VP16 peptide in the cell.
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  The more stable the degron protein construct is, the more likely it is that more GFP will be expressed.
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  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.
 +
        </p>
 +
 
 +
  </body>
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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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