Difference between revisions of "Team:Waterloo/Parts"

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 the [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.

Figure 1. The Hsp104 plasmid.

Figure 2. The insertion of a Gal1,10 promoter and CFP fusion into the Hsp104 plasmid.

Hsp104-SC1

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.

Figure 1. The insertion of a Gal1,10 promoter and CFP fusion containing a premature stop codon into the Hsp104 plasmid.

Figure 2. The exact location of the premature stop codon used to make Hsp104-SC1.

Hsp104-SC2

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.

Figure 1. The insertion of a Gal1,10 promoter and CFP fusion containing a premature stop codon into the Hsp104 plasmid.

Figure 2. The exact location of the premature stop codon used to make Hsp104-SC1.

ADH1-GFP

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.

Figure 1. The ADH1 and GFP fusion.

ADH1-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.

Figure 1. The Gal1 and GFP fusion.

Cup1-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.

Figure 1. The Cup1 and GFP fusion.

Gal1-CFP NSC

This biobrick is a fusion of the Gal1,10 promoter, Cyan Fluorescent Protein (CFP), and the chaperone protein Hsp104.

Figure 1. The Gal1, CFP, and Hsp104 fusion without a premature stop codon in GFP.

Gal1-CFP SC1

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.

Figure 1. The Gal1, CFP, and Hsp104 fusion with a premature stop codon (SC1) in GFP.

Gal1-CFP SC2

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.

Figure 1. The Gal1, CFP, and Hsp104 fusion with a premature stop codon (SC2) in GFP.

Hsp104-Gal1

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.

Figure 1. The insertion of a Gal1,10 promoter and CFP fusion into the Hsp104 plasmid.

Hsp104-Cup1

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) except with the Gal1,10 promoter replaced by a Cup1 promoter and CFP replaced by GFP; this experiment was a positive control to show the normal retention of our constructs with Cup1 promoters by cells.

Figure 1. The insertion of a Cup1 promoter and GFP fusion into the Hsp104 plasmid.

pXP218-ADH1

Compared to other model organisms, yeast is underrepresented in the iGEM registries. As our biobrick contribution, we characterized the ADH1 promoter. We also characterized the promoters Gal1 and Cup1 as controls against which we could compare our experiments. A high copy number plasmid was used in this case (compared to the low copy Hsp104 plasmid) to maximize the promoter activity for quantification.

Blank.

Figure 1. The insertion of a ADH1 promoter and GFP fusion into the Hsp104 plasmid.

pXP218-Gal1

Compared to other model organisms, yeast is underrepresented in the iGEM registries. As our biobrick contribution, we characterized the ADH1 promoter. We also characterized the promoters Gal1 and Cup1 as controls against which we could compare our experiments. A high copy number plasmid was used in this case (compared to the low copy Hsp104 plasmid) to maximize the promoter activity for quantification.

Gal1 is a positively regulated promoter found on the Hsp104 plasmid. Characterization was important for us to understand the transcription rates in a normal cell in order to see the metabolic load of expression of additional Hsp104.

Figure 1. The insertion of a Gal1,10 promoter and GFP fusion into the Hsp104 plasmid.

pXP218-Cup1

Compared to other model organisms, yeast is underrepresented in the iGEM registries. As our biobrick contribution, we characterized the ADH1 promoter. We also characterized the promoters Gal1 and Cup1 as controls against which we could compare our experiments. A high copy number plasmid was used in this case (compared to the low copy Hsp104 plasmid) to maximize the promoter activity for quantification.

Cup 1 is a positively regulated promoter (induced with copper) found on the Sup35 plasmid [BB8]. It is less leaky than other inducible promoters which makes it useful for the study of Sup35, allowing us to control for the amount of Sup35 in the cell to see how the PSI+ state causes further misfolding of Sup35. Characterization of this promoter was therefore important for quantification of the prion response.

Figure 1. The insertion of a Cup1 promoter and GFP fusion into the Hsp104 plasmid.

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