Difference between revisions of "Team:NYMU-Taipei/Basic Part"

 
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<h2>Our Favorite Basic Part: <a href="http://parts.igem.org/Part:BBa_K2751015">BBa_K27510015</a>  </h2>
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<h2>Human DKK1 Promoter</h2>
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<h3>Description </h3>
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<p>The human DKK1 promoter is the promoter regulating the human DKK1 gene. The promoter is located on human chromosome 10, and will be activated upon DKK1 gene activation. </p>
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<h3>Design</h3>
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<img src="https://static.igem.org/mediawiki/parts/thumb/7/70/T--NYMU-Taipei--parts-DP1.png/799px-T--NYMU-Taipei--parts-DP1.png" style="width:900px;">
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<img src="https://static.igem.org/mediawiki/parts/3/39/T--NYMU-Taipei--parts-DP2.png" style="width:700px;">
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<h3>Characterization </h3>
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<p>We used PCR technique to amplify a segment of human chromosome 10 (from 52311944 to 52314294). The estimated length of our DKK1 promoter segment is around 2356bp. </p>
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 +
<h3>Functionality Test</h3>
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<p>To test the functionality of the DKK1 promoter, we transfected HEK 293 cells with a plasmid (BBa_K2751024) containing the DKK1 promoter and a mCherry reporter protein. We added various concentrations of testosterone to stimulate the DKK1 promoter. The expression of mCherry was read by IX83 confocal microscope using the same exposure time, and we selected a field full of cells from each condition group to control the number of cells observed. Quantification of cells expressing the mCherry protein was determined via Metamorph multi-wavelength cell scoring. Intensity 10 gray levels above local background were determined as real fluorescence emission.</p>
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<table style="width:90%">
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  <tr>
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    <th>Picture taken under 10x</th>
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    <th>Concentration of Testosterone</th>
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    <th>Number of cells expressing mCherry protein determined by Metamorph</th>
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  </tr>
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  <tr>
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    <td><img src="https://static.igem.org/mediawiki/parts/1/1a/T--NYMU-Taipei--parts-DP4.png" style="width:400px;"></td>
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    <td>0.05ug/mL</td>
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    <td>14</td>
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  </tr>
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  <tr>
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    <td><img src="https://static.igem.org/mediawiki/parts/c/c7/T--NYMU-Taipei--parts-DP5.png" style="width:400px;"></td>
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    <td>0.5ug/mL</td>
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    <td>72</td>
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  </tr>
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<tr>
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    <td><img src="https://static.igem.org/mediawiki/parts/3/37/T--NYMU-Taipei--parts-DP6.png" style="width:400px;"></td>
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    <td>5ug/mL</td>
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    <td>77</td>
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  </tr>
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</table>
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<p>As the concentration of testosterone rises from 0.05ug/mL to 5ug/mL, the number of cells that express mCherry protein also increases. This shows that testosterone level within a range can stimulate the activation of DKK1 promoter, resulting in increasing expression of mCherry protein. </p>
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<div class="paragraphs">
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<h2>List of Basic Parts</h2>
 
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     <td>B<a href="http://parts.igem.org/Part:BBa_K2751007">Ba_K2751007</a></td>
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     <td><a href="http://parts.igem.org/Part:BBa_K2751007">BBa_K2751007</a></td>
 
     <td>pET32a-FPFSkel1 CyPet LRP6BP3</td>
 
     <td>pET32a-FPFSkel1 CyPet LRP6BP3</td>
 
     <td>Plasmid</td>
 
     <td>Plasmid</td>
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     <td><a href="http://parts.igem.org/Part:BBa_K2751026">BBa_K2751026</a></td>
 
     <td><a href="http://parts.igem.org/Part:BBa_K2751026">BBa_K2751026</a></td>
−
     <td>plasmid2</td>
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     <td>plasmid 2</td>
 
     <td>Plasmid</td>
 
     <td>Plasmid</td>
 
     <td>4240</td>
 
     <td>4240</td>
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   <tr>
 
     <td><a href="http://parts.igem.org/Part:BBa_K2751027">BBa_K2751027</a></td>
 
     <td><a href="http://parts.igem.org/Part:BBa_K2751027">BBa_K2751027</a></td>
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     <td>plasmid2s</td>
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     <td>plasmid 2s</td>
 
     <td>Plasmid</td>
 
     <td>Plasmid</td>
 
     <td>4294</td>
 
     <td>4294</td>

Latest revision as of 03:46, 18 October 2018

HAIR TO STAY
TEAM
Team Members
Collaborations
PROJECT
Description
Design
Experiments & Results
Notebook
InterLab
Model
Demonstrate
Improve
Attributions
PARTS
Parts Overview
Basic Parts
Composite Parts
Part Collection
SAFETY
HUMAN PRACTICES
Human Practices
Education & Engagement
AWARDS
Applied Design
Entrepreneurship
Model
JUDGING FORM ⇗



Our Favorite Basic Part: BBa_K27510015

Human DKK1 Promoter

Description

The human DKK1 promoter is the promoter regulating the human DKK1 gene. The promoter is located on human chromosome 10, and will be activated upon DKK1 gene activation.

Design

Characterization

We used PCR technique to amplify a segment of human chromosome 10 (from 52311944 to 52314294). The estimated length of our DKK1 promoter segment is around 2356bp.

Functionality Test

To test the functionality of the DKK1 promoter, we transfected HEK 293 cells with a plasmid (BBa_K2751024) containing the DKK1 promoter and a mCherry reporter protein. We added various concentrations of testosterone to stimulate the DKK1 promoter. The expression of mCherry was read by IX83 confocal microscope using the same exposure time, and we selected a field full of cells from each condition group to control the number of cells observed. Quantification of cells expressing the mCherry protein was determined via Metamorph multi-wavelength cell scoring. Intensity 10 gray levels above local background were determined as real fluorescence emission.

Picture taken under 10x Concentration of Testosterone Number of cells expressing mCherry protein determined by Metamorph
0.05ug/mL 14
0.5ug/mL 72
5ug/mL 77

As the concentration of testosterone rises from 0.05ug/mL to 5ug/mL, the number of cells that express mCherry protein also increases. This shows that testosterone level within a range can stimulate the activation of DKK1 promoter, resulting in increasing expression of mCherry protein.

List of Basic Parts

Part Number Description Type Length(bp)
BBa_K2751015 Human DKK1 promoter Coding 2356
BBa_K2751008 YPet skeleton Reporter 837
BBa_K2751011 mEGFP skel Reporter 746
BBa_K2751000 pET32a-FPFSkel1 Plasmid 5413
BBa_K2751001 pET32a-FPFSkel2 Plasmid 5437
BBa_K2751002 pET32a-FPFSkel1-YPet-Ubc9 Plasmid 6592
BBa_K2751003 pET32a-FPFSkel1 YPet LRP6BP1BP2 Plasmid 7816
BBa_K2751004 pET32a-FPFSkel1 YPet LRP6BP1 Plasmid 6904
BBa_K2751005 pET32a-FPFSkel1 CyPet SUMO1 Plasmid 6421
BBa_K2751006 pET32a-FPFSkel1 CyPet LRP6BP3BP4 Plasmid 7821
BBa_K2751007 pET32a-FPFSkel1 CyPet LRP6BP3 Plasmid 6892
BBa_K2751024 plasmid 1 Plasmid 5964
BBa_K2751025 plasmid 1s Plasmid 6018
BBa_K2751026 plasmid 2 Plasmid 4240
BBa_K2751027 plasmid 2s Plasmid 4294
BBa_K2751016 pCMV promoter Coding 290
BBa_K2751017 LRP6BP1 Coding 840
BBa_K2751018 Ubc9 Coding 528
BBa_K2751019 mEGFP Coding 720
BBa_K2751020 mEGFP suffix Coding 26
BBa_K2751021 ALB tag Coding 56
BBa_K2751022 YPet Coding 777
BBa_K2751023 mCherry suffix Coding 23