Team:NTNU-Trondheim/Achievements

From 2013.igem.org

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<p style="text-align:center; color:black; "> <b>BRONZE </b></p> </div>
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<p style="text-align:center; color:black; "> <b> Achievements</b></p> </div>
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<p style="text-align:center; color:black; "> Gibson Assembly and transformation</p> </div>
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Our overlapping DNA fragments; tat, GFP, RFP and plasmid backbone was cloned together by <a href="https://2013.igem.org/Team:NTNU-Trondheim/Protocols#Gibson_Assembly"> Gibson Assembly</a> and <a href="https://2013.igem.org/Team:NTNU-Trondheim/Protocols#Transformation"> transformed</a> into <i>E.col</i> strain ER2566 cells. The photograph below shows two of the resulting colonies (named ER1 and ER2) from this transformation.<br><br>
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✔ 1. Team registration<br>
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<div class="col4" style="background-color:white;><a href="https://static.igem.org/mediawiki/2013/b/b0/Colonies.jpg"> <img src="https://static.igem.org/mediawiki/2013/b/b0/Colonies.jpg" width="400">
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<a href="https://igem.org/Team.cgi?year=2013&team_name=NTNU-Trondheim.com">Registered Team</a><br>
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<p style="text-align:center; color:black; "> <b>Figure:</b> Two of the transformed colonies with the tat_GFP_l_RFP construct. Hereby named ER1 and ER2.</p> </div><br><br>
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✔ 2. Complete Judging Form<br>
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✔ 3. Team Wiki<br>
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<a href="https://2013.igem.org/Team:NTNU-Trondheim">NTNU's Wiki Home</a><br>
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✔ 4. Present a poster and a talk at the iGEM Jamboree<br>
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✔ 5. Document at least one new standard BioBrick Part or Device used in your project/central to your project and submit this part to the iGEM Registry (submissions must adhere to the iGEM Registry guidelines). A new application of and outstanding documentation (quantitative data showing the Part’s/ Device’s function) of a previously existing BioBrick part in the “Experience” section of that BioBrick’s Registry entry also counts. Please note you must submit this new part to the iGEM Parts Registry<br>
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We submitted two new characterized BioBrick Parts:<br>
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1. BBa_K1082000 is a Pm/xylS promoter system is positively regulated by m-toluic acid. The m-toluic acid binds to the xylS protein which is constitutively expressed by the Pm/xylS promoter system. The xylS-m-toluic acid complex binds to the Pm promoter, and activates it.<br>
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2. BBa_K1082001 is a composite part that constitutively expresses a tat-GFP-RFP construct. It is a protein coding sequence coding a fusion protein made up by GFP and RFP. In addition, the part contains tat, a signaling sequence responsible for transport to the periplasm.<br>
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<p style="text-align:center; color:black; "><b> SILVER </b></p> </div>
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<p style="text-align:center; color:black; "> Sequencing and characterization</p> </div>
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<p>The plasmid from the ER1 samples was isolated by the <a href="https://2013.igem.org/Team:NTNU-Trondheim/Protocols#Transformed_cells"> Promega Wizard Plus SV Minipreps DNA Purification System A1460</a> and sequenced. Figure below shows the alignment of the sequencing results with the reference DNA sequence.<br>
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✔ 1.Experimentally validate that at least one new BioBrick Part or Device of your own design and construction works as expected.<br>
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<div class="col4" style="background-color:white;><a href="https://static.igem.org/mediawiki/2013/c/cc/ER1.jpg"> <img src="https://static.igem.org/mediawiki/2013/c/cc/ER1.jpg" width="600">
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:/ Pm/XylS ??<br>
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<p style="text-align:center; color:black; "> <b>Figure:</b> Aligment of tat_GFP_l_RFP (ER1) with reference DNA.</p> </div>
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:/ Tat-GFP_RFP doen not work as expected! What can be said about this?<br>
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✔ 2.Document the characterization of this part in the “Main Page” section of that Part’s/Device’s Registry entry.<br>
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<p>The sequence align almost perfectly. There seems to be some sort of extra insert at the linker region, but this insert is dividable by 3, so the reading frame is maintained. This is supported by the fact that the colonies with this construct is red (see figure above).<br>
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1. BBa_K1082000 is a Pm/xylS promoter system.<br>
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Red ER1-cells in liquid media was immobilized in agar and then viewed in a confocal microscope for seeing if RFP was localized in the periplasm. The results can be seen in the two figures below:<br><br>
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:/add something from results here<br>
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2. BBa_K1082001 is a tat-GFP-RFP construct.<br>
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:/add something from results here<br>
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✔ 3.Submit this new part to the iGEM Parts Registry (submissions must adhere to the iGEM Registry guidelines).<br>
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<div class="col4" style="background-color:white;> <a href="https://static.igem.org/mediawiki/2013/d/d8/RFP_ER1.jpg"> <img src="https://static.igem.org/mediawiki/2013/d/d8/RFP_ER1.jpg" width="303">
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<p style="text-align:center; color:black; "> </b>Figure:</b>Red ER1-cells viewed in confocal microscope in 2D </p> </div>
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1. <partinfo>BBa_K1082000</partinfo> is a Pm/XylS promoter system.<br>
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2. <partinfo>BBa_K1082001</partinfo> is a tat-GFP-RFP construct.<br>
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✔ 4.Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe one or more ways in which these or other broader implications have been taken into consideration in the design and execution of your project.<br>
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INCERT STUFF FROM VESICLES SAFETY FROM MARIA
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<div class="col4" style="background-color:white;><a href="https://static.igem.org/mediawiki/2013/a/a0/RFP_3D_ER1.jpg"> <img src="https://static.igem.org/mediawiki/2013/a/a0/RFP_3D_ER1.jpg" width="303">
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<p style="text-align:center; color:black; "> </b>Figure:</b>Red ER1-cells viewed in confocal microscope in 3D </p> </div>
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<p style="text-align:center; color:black; "><b> GOLD</b> </p> </div>
 
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✔ 1. Your project may have implications for the environment, security, safety and ethics and/or ownership and sharing. Describe a novel approach that your team has used to help you and others consider these aspects of the design and outcomes of synthetic biology efforts. Please justify its novelty and how this approach might be adapted and scaled for others to use.<br>
 
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INCERT MARIAS STUFF ON VESICLES.
 

Revision as of 16:12, 4 October 2013

Trondheim iGEM 2013

header
Mercury
Achievements



Gibson Assembly and transformation



Our overlapping DNA fragments; tat, GFP, RFP and plasmid backbone was cloned together by Gibson Assembly and transformed into E.col strain ER2566 cells. The photograph below shows two of the resulting colonies (named ER1 and ER2) from this transformation.

Figure: Two of the transformed colonies with the tat_GFP_l_RFP construct. Hereby named ER1 and ER2.



Sequencing and characterization



The plasmid from the ER1 samples was isolated by the Promega Wizard Plus SV Minipreps DNA Purification System A1460 and sequenced. Figure below shows the alignment of the sequencing results with the reference DNA sequence.

Figure: Aligment of tat_GFP_l_RFP (ER1) with reference DNA.


The sequence align almost perfectly. There seems to be some sort of extra insert at the linker region, but this insert is dividable by 3, so the reading frame is maintained. This is supported by the fact that the colonies with this construct is red (see figure above).
Red ER1-cells in liquid media was immobilized in agar and then viewed in a confocal microscope for seeing if RFP was localized in the periplasm. The results can be seen in the two figures below:

Figure:Red ER1-cells viewed in confocal microscope in 2D


Figure:Red ER1-cells viewed in confocal microscope in 3D


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