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> | + | <style> |
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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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| − | <img src="https://static.igem.org/mediawiki/parts/7/75/Nymu-fyve.jpg" style="padding- | + | <div class="item" style="padding-left:16%;width:22%"> |
| − | < | + | <img src="https://static.igem.org/mediawiki/parts/7/75/Nymu-fyve.jpg" style="padding-bottom:2%;width:100%"> |
| − | </ | + | <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=" | + | <img src="https://static.igem.org/mediawiki/parts/1/1b/Nymu-fyve-wort.jpg" style="padding-bottom:2%;width:100%"> |
| − | </ | + | <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
This experience page is provided so that any user may enter their experience using this part.
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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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