Difference between revisions of "Team:BIT-China/Project/Detection"
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<h2 class="title-h2">Detection</h2> | <h2 class="title-h2">Detection</h2> | ||
<div class="vs-content"> | <div class="vs-content"> | ||
| − | <h3 class="title-h3"> | + | <h3 class="title-h3">the detection circuit </h3> |
| − | <p class="my-content-p">To measure the sweetness of sweeteners, we | + | <p class="my-content-p">To measure the sweetness of sweeteners, we designed the detection device consisting of the promoter Pfus, the reporter gene <i>mRFP</i>, and the terminator <i>CYC1t</i>. </p> |
<div class="my-img-box"> | <div class="my-img-box"> | ||
<img src="https://static.igem.org/mediawiki/2017/4/45/T--BIT-China--2017project_detection.png" alt=""> | <img src="https://static.igem.org/mediawiki/2017/4/45/T--BIT-China--2017project_detection.png" alt=""> | ||
</div> | </div> | ||
| − | <p class="my-content-p"> | + | <p class="my-content-p">The upstream signal will be produced when the human sweet taste receptor T1R2-T1R3 detects sweeteners, the signal can be transmitted to detection device through the MAPK pathway which exists in yeast naturally. And this signal will activate the promoter <i>P<sub>fus</sub></i> specificitly, thereby initiating the expression of the <i>mRFP</i> reporter gene. |
| + | </p> | ||
| − | <p class="my-content-p"> In order to construct | + | |
| + | <p class="my-content-p">In order to construct the detection circuit, firstly, we connected the three parts <i>P<sub>fus</sub></i> , <i>mRFP</i>, and <i>CYC1t</i> by OE-PCR. However, the results were not as good as we expected. So we changed the method to Gibson assembly to connect this device with the linear plasmid pRS42K. </p> | ||
| − | + | <div class="my-img-box"> | |
| + | <img style="width: 60%; height: 60%" src="https://2017.igem.org/File:T-BIT-China-2017yhy-19.png" alt=""> | ||
| + | |||
| + | <p style="text-align: center">Fig.2 construction of detection circuit by Gibson assembly</p> | ||
| + | |||
| + | </div> | ||
<p class="my-content-li2">Group A: transformated CENPK2-1C(a type) and CENPK2-1D(a type)</p> | <p class="my-content-li2">Group A: transformated CENPK2-1C(a type) and CENPK2-1D(a type)</p> | ||
Revision as of 09:33, 27 October 2017
Detection
the detection circuit
To measure the sweetness of sweeteners, we designed the detection device consisting of the promoter Pfus, the reporter gene mRFP, and the terminator CYC1t.
The upstream signal will be produced when the human sweet taste receptor T1R2-T1R3 detects sweeteners, the signal can be transmitted to detection device through the MAPK pathway which exists in yeast naturally. And this signal will activate the promoter Pfus specificitly, thereby initiating the expression of the mRFP reporter gene.
In order to construct the detection circuit, firstly, we connected the three parts Pfus , mRFP, and CYC1t by OE-PCR. However, the results were not as good as we expected. So we changed the method to Gibson assembly to connect this device with the linear plasmid pRS42K.
Fig.2 construction of detection circuit by Gibson assembly
Group A: transformated CENPK2-1C(a type) and CENPK2-1D(a type)
Group B: transformated CENPK2-1C(a type) alone
Group C: CENPK2-1D(a type) alone
After 9 hours, group A was fluorescent, and group B and group C didn’t fluoresce. The result means signal reporter device worked.
