Team:Washington/Our Project

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  <h1> METHODS </h1>
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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>
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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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<h2>Cloning in <i>E.Coli </i> </h2>
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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
 +
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
 +
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
 +
measurements conducted on each cell culture.</p> 
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</body>
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</html>

Revision as of 01:21, 8 October 2014

UW Homepage Official iGEM website

METHODS

The essential process of our system involves cloning and manufacturing of a plasmid in E.Coli . Once, the plasmids have been constructed and verified in E.Coli they are transformed into S.Cerevisiae. The plasmid constructs are then expressed. Following several days of growth the yeast cultures are passed through a flow cytometer 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 stability.

Cloning in E.Coli

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 it.

Preparation and Passaging of Saccharomyces cerevisiae

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 S. Cerevisiae with the ability to produce green fluorescent proteins with the proper promoter protein such as Gal4 which is a part of our degron construct. Following the transformation, the cells are plated onto plates with on a Selective Dropout C-Uracil media and incubated at 30oC for 2 days. After two days, three 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 30oC. 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 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 culture and flow cytometry is run on the culture.

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 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 measurements conducted on each cell culture.

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