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| | <h1> Background </h1> | | <h1> Background </h1> |
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| - | <p> 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 choosen. 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.
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| | <h2> Our System </h2> | | <h2> Our System </h2> |
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| | <h2> Method </h2> | | <h2> Method </h2> |
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| - | <p> 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>
| + | <h3> Analysis of Test Protein Stability </h3> |
| - | 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
| + | <h3> Cloning in <i> Escherichia coli </i> </h3> |
| - | stability. </p>
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| - | | + | <h3> Preparation and Passaging of <i> Saccharomyces cerevisiae </i> </h3> |
| - | <h3> Stability of the Test Protein analyzed via Circular Dichroism Thermalmelts and Guanadine Hcl Metls </h3>
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| - | <h3> Cloning in <i>E.Coli </i> </h3>
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| - | <p> 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> 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 choosen and added to an overnight culture of 3mL Selective Dropout Media C-Uracil and 2% Glucose then incuabted 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> 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> | | <h3> Relative Stability Analyzed via Flow Cytometry </h3> |
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| | <h3> Mutagenesis through Error Prone Polymerase Chain Reactions </h3> | | <h3> Mutagenesis through Error Prone Polymerase Chain Reactions </h3> |
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| | <h2> Results </h2> | | <h2> Results </h2> |
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| | + | <h3> Protein stability analysis using Thermal and Guanadine-HCl melts </h3> |
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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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| | <h2> Future Plans </h2> | | <h2> Future Plans </h2> |
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| | + | <h3> Examination of more proteins </h3> |
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| | <h2> Submitted Parts </h2> | | <h2> Submitted Parts </h2> |
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