Difference between revisions of "Part:BBa K2368006"
| (22 intermediate revisions by 2 users not shown) | |||
| Line 6: | Line 6: | ||
<h3>General</h3> | <h3>General</h3> | ||
| − | <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 | + | <p>To measure the sweetness of sweeteners, we designed the detection circuit. This composite part is based on the <i>Saccharomyces cerevisiae</i> pheromone signaling pathway. It contains a yeast induced promoter <i>P<sub>fus</sub></i> , a <i>mRFP </i> reporter, and a yeast terminator. In <i>CEN.PK2-1C </i> (a mating type yeast) <i>P<sub>fus</sub></i> is activated by α pheromone, thereby initiating the expression of the reporter gene.</p> |
| − | + | [[File:T_BIT-China_2017part_8.png|center|500px|默认文字]] | |
| − | <p style="text-align: center"> | + | <p style="text-align: center">Fig.1 The detection circuit</p> |
| − | <p>We constructed | + | <p>We constructed <i>P<sub>fus</sub></i>-<i>mRFP-cyc1t-PRS42K</i> recombinant plasmid, and transformed it into <i>CEN.PK2-1C</i> (a type). </p> |
| − | + | [[File:T_BIT-China_2017part_9.png|center|300px|默认文字]] | |
| − | + | <p style="text-align: center">Fig.2 The result of colony PCR</p> | |
| − | <p style="text-align: center"> | + | |
<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> | ||
| − | < | + | |
| − | <p style="text-align: center">a</p> | + | <div style="margin-left: 270px"><ul> |
| + | <li style="display: inline-block;"> [[File:T_BIT-China_2017part_10.png|center|center|220px|a]] </li> | ||
| + | <li style="display: inline-block;"> [[File:T_BIT-China_2017part_11.png|center|center|220px|b]] </li> | ||
| + | </ul></div> | ||
| + | <p style="text-align: center">Fig.3 Images of yeast fluorescence. a: Initial fluorescence image; b: Fluorescence image after 4 hours.</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> | <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 style="text-align: center"> | + | [[File:T_BIT-China_2017part_12.png|center|500px|默认文字]] |
| + | <p style="text-align: center">Fig.4 <i>mRFP</i> intensity of <i>CEN.PK2-1C</i> 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> | ||
| Line 28: | Line 32: | ||
<!-- --> | <!-- --> | ||
<h2>Sequence and Features</h2> | <h2>Sequence and Features</h2> | ||
| − | <partinfo>BBa_K2368006 SequenceAndFeatures | + | <partinfo>BBa_K2368006 SequenceAndFeatures |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
Latest revision as of 07:21, 24 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 colony PCR
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.
Fig.3 Images of yeast fluorescence. a: Initial fluorescence image; b: Fluorescence image after 4 hours.
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 mRFP 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]