Team:Hannover/Results/Heavy Metals/Arabidopsis

From 2014.igem.org

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<h2>Results</h2>
<h2>Results</h2>
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<p class="text">The exchange of the promoter was checked by sequencing. We achieved to regenerate young <i>A. thaliana</i> after the transformation with our T4MBP.</p>
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<p class="text">The exchange of the promoter was checked by sequencing. We achieved to regenerate young <i>A. thaliana</i> after the transformation with our T4MBP. To receive a stabile F2 generation seeds of transformed plants would have to be harvested and grown. As a next step of this test series the plants would have been transfered on medium with heavy metals. In a following analysis it would be detected if these plants bind the heavy metal zinc, copper, cadmium and/or arsenic.    </p>
<center><table border="0"><tr><td><a href="https://static.igem.org/mediawiki/2014/6/6a/Hannover_20141012_Arabudopsis-results1.jpg" data-lightbox="galery1" data-title="Fig. 1a: Young potentially transgenic <i>A. thaliana</i> during the transfer from medium to earth. "><img src="https://static.igem.org/mediawiki/2014/6/6a/Hannover_20141012_Arabudopsis-results1.jpg" width="300px"></a></td><td><a href="https://static.igem.org/mediawiki/2014/1/13/Hannover_20141012_Arabudopsis-results3.jpg
<center><table border="0"><tr><td><a href="https://static.igem.org/mediawiki/2014/6/6a/Hannover_20141012_Arabudopsis-results1.jpg" data-lightbox="galery1" data-title="Fig. 1a: Young potentially transgenic <i>A. thaliana</i> during the transfer from medium to earth. "><img src="https://static.igem.org/mediawiki/2014/6/6a/Hannover_20141012_Arabudopsis-results1.jpg" width="300px"></a></td><td><a href="https://static.igem.org/mediawiki/2014/1/13/Hannover_20141012_Arabudopsis-results3.jpg
" data-lightbox="galery1" data-title="Fig. 1b: Young potentially transgenic <i>A. thaliana</i> in medium."><img src="https://static.igem.org/mediawiki/2014/1/13/Hannover_20141012_Arabudopsis-results3.jpg" width="330px"></a></td><td><a href="https://static.igem.org/mediawiki/2014/3/35/Hannover_20141012_Arabudopsis-results2.jpg
" data-lightbox="galery1" data-title="Fig. 1b: Young potentially transgenic <i>A. thaliana</i> in medium."><img src="https://static.igem.org/mediawiki/2014/1/13/Hannover_20141012_Arabudopsis-results3.jpg" width="330px"></a></td><td><a href="https://static.igem.org/mediawiki/2014/3/35/Hannover_20141012_Arabudopsis-results2.jpg
" data-lightbox="galery1" data-title="Fig. 1c:Young potentially transgenic <i>A. thaliana</i> in earth."><img src="https://static.igem.org/mediawiki/2014/3/35/Hannover_20141012_Arabudopsis-results2.jpg" width="330px"></a></td>
" data-lightbox="galery1" data-title="Fig. 1c:Young potentially transgenic <i>A. thaliana</i> in earth."><img src="https://static.igem.org/mediawiki/2014/3/35/Hannover_20141012_Arabudopsis-results2.jpg" width="330px"></a></td>
</tr><tr><th colspan="3"><p class="text">Fig. 1: Pictures showing young potentially transgenic <i>A. thaliana</i> before, during and after the transfer from medium to earth. </th></p></td></tr></table></center>
</tr><tr><th colspan="3"><p class="text">Fig. 1: Pictures showing young potentially transgenic <i>A. thaliana</i> before, during and after the transfer from medium to earth. </th></p></td></tr></table></center>
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<p class="text">Below in figures 2 and 3 you can see the original vector pORE_E3 with an enTCUP3 promoter. For a better expression of our T4MBP protein we exchanged the enTCUP3 with the 2x35S promoter. Each step of this procedure is visualized in <a href="https://static.igem.org/mediawiki/2014/d/d1/Hannover_20141015_PORE_E3_2x35S%2BInsert_Anke_Fabian.dna_History.png">this histroy </a>. Furthermore this history includes the insertion of our T4MBP (there termed CDS for coding sequence). </p>
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<center><table border="0">
<center><table border="0">
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<tr><td><a href="https://static.igem.org/mediawiki/2014/9/97/Hannover_20141015_PORE_E3_2x35S_T4MBP_Map-1.png" data-lightbox="galery1" data-title="Fig. 2a: pORE-E3 vector with 2x35S promoter"><img src="https://static.igem.org/mediawiki/2014/9/97/Hannover_20141015_PORE_E3_2x35S_T4MBP_Map-1.png" width="300px"></a></td>
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<td><a href="https://static.igem.org/mediawiki/2014/d/d1/Hannover_20141015_PORE_E3_2x35S%2BInsert_Anke_Fabian.dna_History.png" data-lightbox="galery1" data-title="Fig. 2b: detailed history of exchanging the enTCUP2 promoter with the 2x35S promoter."><img src="https://static.igem.org/mediawiki/2014/d/d1/Hannover_20141015_PORE_E3_2x35S%2BInsert_Anke_Fabian.dna_History.png" width="300px"></td></tr>
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<col width=500>
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<tr><th colspan="3"><p class="text">Fig. 2: Pictures showing the vector pORE_E3 with 2x35S promoter and the way of exchanging the enTCUP2 promoter with the 2x35S promoter. Each step that have been done to exchange these promoters is shwon in <a href="https://static.igem.org/mediawiki/2014/d/d1/Hannover_20141015_PORE_E3_2x35S%2BInsert_Anke_Fabian.dna_History.png">this histroy </a>.</th></p></td></tr></table></center>
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<col width=500>
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<tr><td><a href="https://static.igem.org/mediawiki/2014/5/5a/Hannover_20141016_PORE-E3_AY562536_Map.png" data-lightbox="galery1" data-title="Fig. 2: Vector pORE_E3 with the original enTCUP2 promoter."><img src="https://static.igem.org/mediawiki/2014/5/5a/Hannover_20141016_PORE-E3_AY562536_Map.png" width="300px"></td><td><a href="https://static.igem.org/mediawiki/2014/9/97/Hannover_20141015_PORE_E3_2x35S_T4MBP_Map-1.png" data-lightbox="galery1" data-title="Fig. 3: Vector pORE-E3 with 2x35S promoter and our T4MBP which includes a sequence for expansin 4, cellulose-binding domain and domains for the binding of copper, arsenic, zinc, cadmium. "><img src="https://static.igem.org/mediawiki/2014/9/97/Hannover_20141015_PORE_E3_2x35S_T4MBP_Map-1.png" width="300px"></a></td></tr>
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<tr><td><p class="text">Fig. 2: Vector pORE_E3 with the original enTCUP2 promoter. </td><td>Fig. 3: Vector pORE-E3 with 2x35S promoter and our T4MBP which includes a sequence for expansin 4, cellulose-binding domain and domains for the binding of copper, arsenic, zinc, cadmium.</p></td></tr></table></center>
</table>
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Revision as of 19:34, 16 October 2014

Results / Heavy metals / Arabidopsis thaliana T4MBP

Labwork

Transformation of A. thaliana with our T4MBP:

  1. Exchange the original promoter enTCUP2 of the binary vector pORE_E3 (AY562536.1) into a 2x35s promoter using resctriction site based cloning.
  2. We integrated our T4MBP in our modified pORE_E3_2x35S.
  3. Application of our floral dip method.
  4. After 6 weeks we harvested transgenic seeds from A. thaliana and plated these seeds on selection MSO-media.
  5. At last we potted transformed plants.

Results

The exchange of the promoter was checked by sequencing. We achieved to regenerate young A. thaliana after the transformation with our T4MBP. To receive a stabile F2 generation seeds of transformed plants would have to be harvested and grown. As a next step of this test series the plants would have been transfered on medium with heavy metals. In a following analysis it would be detected if these plants bind the heavy metal zinc, copper, cadmium and/or arsenic.

Fig. 1: Pictures showing young potentially transgenic A. thaliana before, during and after the transfer from medium to earth.


Below in figures 2 and 3 you can see the original vector pORE_E3 with an enTCUP3 promoter. For a better expression of our T4MBP protein we exchanged the enTCUP3 with the 2x35S promoter. Each step of this procedure is visualized in this histroy . Furthermore this history includes the insertion of our T4MBP (there termed CDS for coding sequence).

Fig. 2: Vector pORE_E3 with the original enTCUP2 promoter.

Fig. 3: Vector pORE-E3 with 2x35S promoter and our T4MBP which includes a sequence for expansin 4, cellulose-binding domain and domains for the binding of copper, arsenic, zinc, cadmium.





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