Difference between revisions of "Part:BBa K2368006"
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<p>To measure the sweetness of sweeteners, we designed the detection circuit. This composite part is based on the Saccharomyces cerevisiae pheromone signaling pathway. It contains a yeast induced promoter Pfus, a mRFP reporter, and a yeast terminator. In CEN.PK2-1C (a mating type yeast) Pfus is activated by α pheromone, thereby initiating the expression of the reporter gene.</p> | <p>To measure the sweetness of sweeteners, we designed the detection circuit. This composite part is based on the Saccharomyces cerevisiae pheromone signaling pathway. It contains a yeast induced promoter Pfus, a mRFP reporter, and a yeast terminator. In CEN.PK2-1C (a mating type yeast) Pfus is activated by α pheromone, thereby initiating the expression of the reporter gene.</p> | ||
| − | [[File:T_BIT-China_2017part_8.png|center| | + | [[File:T_BIT-China_2017part_8.png|center|500px|默认文字]] |
<p style="text-align: center">fig.1 The detection circuit</p> | <p style="text-align: center">fig.1 The detection circuit</p> | ||
<p>We constructed Pfus-mRFP-cyc1t-PRS42K recombinant plasmid, and transformed it into CEN.PK2-1C (a type). </p> | <p>We constructed Pfus-mRFP-cyc1t-PRS42K recombinant plasmid, and transformed it into CEN.PK2-1C (a type). </p> | ||
| − | [[File:T_BIT-China_2017part_9.png|center| | + | [[File:T_BIT-China_2017part_9.png|center|300px|默认文字]] |
<p style="text-align: center">fig.2 The result of cPCR</p> | <p style="text-align: center">fig.2 The result of cPCR</p> | ||
<p>Cells transformed with the signal reporter device were cultured to the exponential phase and treated with α pheromone.</p> | <p>Cells transformed with the signal reporter device were cultured to the exponential phase and treated with α pheromone.</p> | ||
<p>We added 5umol/L α pheromone into the transformed yeast. And after 4 hours, it was fluorescent.</p> | <p>We added 5umol/L α pheromone into the transformed yeast. And after 4 hours, it was fluorescent.</p> | ||
| − | < | + | |
| − | T_BIT-China_2017part_10.png|a | + | <div><ul> |
| − | T_BIT-China_2017part_11.png|b | + | <li style="display: inline-block;"> [[File:T_BIT-China_2017part_10.png|thumb|none|220px|a]] </li> |
| − | </ | + | <li style="display: inline-block;"> [[File:T_BIT-China_2017part_11.png|thumb|none|220px|b]] </li> |
| + | </ul></div> | ||
<p>In order to characterize the function of detection circuit, we measured absorbance and fluorescence of the cells in the plate reader after treating them with varying concentrations of α pheromone. </p> | <p>In order to characterize the function of detection circuit, we measured absorbance and fluorescence of the cells in the plate reader after treating them with varying concentrations of α pheromone. </p> | ||
| − | [[File:T_BIT-China_2017part_12.png|center| | + | [[File:T_BIT-China_2017part_12.png|center|500px|默认文字]] |
<p style="text-align: center">fig.4 RFP intensity of CEN.PK2-1C induced by α factor</p> | <p style="text-align: center">fig.4 RFP intensity of CEN.PK2-1C induced by α factor</p> | ||
<p>As shown in this figure, adding higher concentration of α pheromone, the fluorescence intensity of the yeast increases. After 20 hours, α pheromone gradually failed, and the fluorescence intensity also decreased.</p> | <p>As shown in this figure, adding higher concentration of α pheromone, the fluorescence intensity of the yeast increases. After 20 hours, α pheromone gradually failed, and the fluorescence intensity also decreased.</p> | ||
Revision as of 12:43, 21 October 2017
Introduction
Signal reporter device (Pfus-mRFP-CYC1t)
General
To measure the sweetness of sweeteners, we designed the detection circuit. This composite part is based on the Saccharomyces cerevisiae pheromone signaling pathway. It contains a yeast induced promoter Pfus, a mRFP reporter, and a yeast terminator. In CEN.PK2-1C (a mating type yeast) Pfus is activated by α pheromone, thereby initiating the expression of the reporter gene.
fig.1 The detection circuit
We constructed Pfus-mRFP-cyc1t-PRS42K recombinant plasmid, and transformed it into CEN.PK2-1C (a type).
fig.2 The result of cPCR
Cells transformed with the signal reporter device were cultured to the exponential phase and treated with α pheromone.
We added 5umol/L α pheromone into the transformed yeast. And after 4 hours, it was fluorescent.
In order to characterize the function of detection circuit, we measured absorbance and fluorescence of the cells in the plate reader after treating them with varying concentrations of α pheromone.
fig.4 RFP intensity of CEN.PK2-1C induced by α factor
As shown in this figure, adding higher concentration of α pheromone, the fluorescence intensity of the yeast increases. After 20 hours, α pheromone gradually failed, and the fluorescence intensity also decreased.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 993
Illegal AgeI site found at 1105 - 1000COMPATIBLE WITH RFC[1000]