Difference between revisions of "Part:BBa K1769000:Experience"
 
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<p>We test this part in BY-2 tobacoo cell line. First we construct this part in vector PSAT6. It has a promoter CAMV35S that act as a consitiutive promoter in plat cells. To see if the dimeric FYVE protein domain really binds to PI3P receptor on the endosome of plant cell, we added a Phosphoinositide-3-kinase(PI3K) inhibitor, wortmannin, that can inhibit PI3K from turning PI3P precursor into PI3P.</p>
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<p>We test this part in BY-2 tobacoo cell line. BY-2 tobacoo cell is a protoplast that is much easier for us to work with compared to agrobacterium-mediated transfection. First we construct this part in vector PSAT6. It has a promoter CAMV35S that act as a consitiutive promoter in plat cells. To see if the dimeric FYVE protein domain really binds to PI3P receptor on the endosome of plant cell, we added a Phosphoinositide-3-kinase(PI3K) inhibitor, wortmannin, that can inhibit PI3K from turning PI3P precursor into PI3P.</p>
 
<p>The picture on the left shows that after we introduce dimeric FYVE into BY-2 tobacoo cell line, the FYVE concentrated in endosome and cell membrane. However, one hour after we added 10<sup>-6</sup>M wortmannin, dimeric FYVE redistributed in cytoplasm.</p> <br>
 
<p>The picture on the left shows that after we introduce dimeric FYVE into BY-2 tobacoo cell line, the FYVE concentrated in endosome and cell membrane. However, one hour after we added 10<sup>-6</sup>M wortmannin, dimeric FYVE redistributed in cytoplasm.</p> <br>
  
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<div class="item" style="padding-left:16%;width:22%">
 
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     <img src="https://static.igem.org/mediawiki/parts/7/75/Nymu-fyve.jpg" style="padding-bottom:2%;width:100%">
 
     <img src="https://static.igem.org/mediawiki/parts/7/75/Nymu-fyve.jpg" style="padding-bottom:2%;width:100%">
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     <span class="caption">Oomycete and potato cell</span>
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     <span class="caption">Fig1. Cells that produces dimeric FYVE and GFP fusion protein. In this picture, the dimeric FYVE binds to the cell membrane and endosome.</span>
 
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     <img src="https://static.igem.org/mediawiki/parts/1/1b/Nymu-fyve-wort.jpg" style="padding-bottom:2%;width:100%">
 
     <img src="https://static.igem.org/mediawiki/parts/1/1b/Nymu-fyve-wort.jpg" style="padding-bottom:2%;width:100%">
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     <span class="caption">Effector protein bonding with PI3P and entering</span>
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     <span class="caption">Fig2. After we added wortmannin the dimeric FYVE and GFP fusion protein redistributed in the cell</span>
 
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Latest revision as of 01:24, 18 September 2015

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Please enter how you used this part and how it worked out.

Applications of BBa_K1769000

Results

We test this part in BY-2 tobacoo cell line. BY-2 tobacoo cell is a protoplast that is much easier for us to work with compared to agrobacterium-mediated transfection. First we construct this part in vector PSAT6. It has a promoter CAMV35S that act as a consitiutive promoter in plat cells. To see if the dimeric FYVE protein domain really binds to PI3P receptor on the endosome of plant cell, we added a Phosphoinositide-3-kinase(PI3K) inhibitor, wortmannin, that can inhibit PI3K from turning PI3P precursor into PI3P.

The picture on the left shows that after we introduce dimeric FYVE into BY-2 tobacoo cell line, the FYVE concentrated in endosome and cell membrane. However, one hour after we added 10-6M wortmannin, dimeric FYVE redistributed in cytoplasm.


Fig1. Cells that produces dimeric FYVE and GFP fusion protein. In this picture, the dimeric FYVE binds to the cell membrane and endosome.
Fig2. After we added wortmannin the dimeric FYVE and GFP fusion protein redistributed in the cell


User Reviews

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