Difference between revisions of "Team:Waterloo/Parts"
| Line 154: | Line 154: | ||
</div> | </div> | ||
<div class="wcontent_alt"> | <div class="wcontent_alt"> | ||
| − | <a href="" ng-click="active='Gal'">Hsp104-Gal1</a> | + | <a href="" ng-click="active='Gal'">Hsp104-Gal1 (Gene Retention)</a> |
</div> | </div> | ||
<div class="wcontent_alt"> | <div class="wcontent_alt"> | ||
| − | <a href="" ng-click="active='Cup'">Hsp104-Cup1</a> | + | <a href="" ng-click="active='Cup'">Hsp104-Cup1 (Gene Retention)</a> |
</div> | </div> | ||
<div class="wcontent_alt"> | <div class="wcontent_alt"> | ||
| Line 170: | Line 170: | ||
</div> | </div> | ||
</div> | </div> | ||
| + | |||
| + | <!-- Our System --> | ||
<div class="col-lg-8"> | <div class="col-lg-8"> | ||
<div class="wcontent-box" id="NSC" ng-show="active == 'NSC'"> | <div class="wcontent-box" id="NSC" ng-show="active == 'NSC'"> | ||
| Line 224: | Line 226: | ||
</div> | </div> | ||
</div> | </div> | ||
| + | |||
| + | <!-- Biobricks --> | ||
<div class="col-lg-8"> | <div class="col-lg-8"> | ||
<div class="wcontent-box" id="ADH1-GFP" ng-show="active == 'ADH1-GFP'"> | <div class="wcontent-box" id="ADH1-GFP" ng-show="active == 'ADH1-GFP'"> | ||
| Line 304: | Line 308: | ||
<div class="wcontent-caption paragraph-medium"> | <div class="wcontent-caption paragraph-medium"> | ||
The Gal1, CFP, and Hsp104 fusion with a premature stop codon (SC2) in GFP. | The Gal1, CFP, and Hsp104 fusion with a premature stop codon (SC2) in GFP. | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | <!-- Promoter Testing --> | ||
| + | <div class="col-lg-8"> | ||
| + | <div class="wcontent-box" id="Gal" ng-show="active == 'Gal'"> | ||
| + | <div class="wcontent-title"> | ||
| + | Hsp104-Gal1 | ||
| + | </div> | ||
| + | <div class="wcontent"> | ||
| + | <p class="paragraph-medium">The purpose of our gene retention experiments was to observe the retention of our plasmid by the yeast cells. We used GFP-tagged Hsp104 under the influence of different promoters to study the plasmid retention. A low copy number plasmid was used to more accurately study the partitioning of plasmids into daughter cells. It was also important that we use the same plasmid as our working prototype so that the findings from this experiment could be directly applied to our other data. </p> | ||
| + | <p class="paragraph-medium">This construct is the same one used in the prototype (NSC); this experiment was a positive control to show the normal retention of our construct by cells. </p> | ||
| + | <img src="https://static.igem.org/mediawiki/2016/a/aa/T--Waterloo--HSP104_LEU_Addgene_Annotate_and_CFP_Gal1.png" class="wcontent-img-solo37"> | ||
| + | <div class="wcontent-caption paragraph-medium"> | ||
| + | The insertion of a Gal1,10 promoter and CFP fusion into the Hsp104 plasmid. | ||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 21:02, 15 October 2016
This construct is a control to test the metabolic load of the Hsp104-CFP fusion protein and as a baseline against which we compare the change in [PSI+]/[psi-] state. We synthesized a fragment to clone into the [Hsp-PRS315]. This plasmid will be referred to as the Hsp104 plasmid and is used in several of our experiments.
CFP was synthesized with a stop codon in the place of Tyr-39 (TAC -> TAG) and amplified with flanking ApaI and BamHI sites. The premature stop codon (before the CFP fluorophore) was expected to truncate the protein during normal transcription. Fluorimetry readings were then used to quantify the amount of read-through for the CFP-tagged Hsp104.
CFP was synthesized with a stop codon in the place of Val-22 (TCA -> TAG) and amplified with flanking ApaI and BamHI sites. The premature stop codon (before the CFP fluorophore) was expected to truncate the protein during normal transcription. Fluorimetry readings were then used to quantify the amount of read-through for the CFP-tagged Hsp104.
This biobrick contains a fusion of the ADH1 promoter and GFP. ADH1 is a eukaryotic promoter; the presence of ethanol induces it. The fusion between it and GFP allows the strength of the promoter to be quantized in varying concentrations of ethanol using the fluorescence of the GFP.
This biobrick is a fusion of the Gal1 promoter and GFP protein. Gal1 is a eukaryotic promoter; the presence of galactose induces it. The fusion allows the strength of the promoter to be quantized in the presence of varying concentrations of galactose using the fluorescence of the GFP.
This biobrick is a fusion of the Cup1 promoter and GFP protein. Cup1 is a eukaryotic promoter; the presence of Cu2+ induces it. The fusion allows the strength of the promoter to be quantized in the presence of varying concentrations of copper ions using the fluorescence of the GFP.
This biobrick is a fusion of the Gal1,10 promoter, Cyan Fluorescent Protein (CFP), and the chaperone protein Hsp104.
This biobrick is a fusion of the Gal1,10 promoter, Cyan Fluorescent Protein (CFP), and the chaperone protein Hsp104 with a premature stop codon introduced at position 1.
This biobrick is a fusion of the Gal1,10 promoter, Cyan Fluorescent Protein (CFP), and the chaperone protein Hsp104 with a premature stop codon introduced at position 2.
The purpose of our gene retention experiments was to observe the retention of our plasmid by the yeast cells. We used GFP-tagged Hsp104 under the influence of different promoters to study the plasmid retention. A low copy number plasmid was used to more accurately study the partitioning of plasmids into daughter cells. It was also important that we use the same plasmid as our working prototype so that the findings from this experiment could be directly applied to our other data.
This construct is the same one used in the prototype (NSC); this experiment was a positive control to show the normal retention of our construct by cells.
