Team:NTNU-Trondheim/Achievements

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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; "> BRONZE </p> </div>
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<p style="text-align:center; color:black; "><b> Gibson Assembly and transformation</b></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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<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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[https://igem.org/Team.cgi?year=2013&team_name=NTNU-Trondheim.com Registered Team]<br>
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✔ 2. Complete Judging Form<br>
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<p style="text-align:center; color:black; "><b> Sequencing and characterization</b></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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<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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<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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[https://2013.igem.org/Team:NTNU-Trondheim NTNU's Wiki Home]<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). Sequencing confirms that we have succesfully made our tat_GFP_RFP construct.<br>
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✔ 4. Present a poster and a talk at the iGEM Jamboree<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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✔ 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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<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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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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<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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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> Pm/xylS promoter </b></p> </div>
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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.
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The Pm/xylS promoter was modified (removing an XbaI site) and turned into a BioBrick by adding the prefix and suffix. With 3A assembly, the promoter was attached to a GFP generator (BBa_E0240), and a backbone ( pSB3K3).
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:/ Pm/XylS ??
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The results show that the GFP are expressed in the samples with the Pm/xylS promoter, indicating that the promoter works. The figures below show the result from the testing. As expected, the reference promoter has a high rate of GFP synthesis.  
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:/ Tat-GFP_RFP doen not work as expected! What can be said about this?
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✔ 2.Document the characterization of this part in the “Main Page” section of that Part’s/Device’s Registry entry.
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1. BBa_K1082000 is a Pm/xylS promoter system.
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<div class="col6" style="background-color:white;><a href="https://static.igem.org/mediawiki/2013/d/dd/Pmxylsprom.png"> <img src="https://static.igem.org/mediawiki/2013/d/dd/Pmxylsprom.png" width="480">
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:/add something from results here
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<p style="text-align:center; color:black; "><b> Figure X:</b> The ratio of GFP synthesis as a function of inducer concentration</p> </div>
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2. BBa_K1082001 is a tat-GFP-RFP construct.
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<div class="col6" style="background-color:white;><a href="https://static.igem.org/mediawiki/2013/2/21/Ref.prom.png"> <img src="https://static.igem.org/mediawiki/2013/2/21/Ref.prom.png" width="480">
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  <p style="text-align:center; color:black; "><b> Figure X:</b> The ratio of GFP synthesis as a function of time</p> </div>
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1. <partinfo>BBa_K1082000</partinfo> is a Pm/XylS promoter system.
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2. <partinfo>BBa_K1082001</partinfo> is a tat-GFP-RFP construct.
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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.
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INCERT STUFF FROM VESICLES SAFETY FROM MARIA
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<p style="text-align:center; color:black; "><b> Protein G</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.
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We have introduced Protein G from <i>Streptococcus dysgalactiae ssp equisimilis</i> into <i>E.coli</i> where it is succesfully expressed.</p><br><br>
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<div class="col4" style="background-color:white;><a href="https://static.igem.org/mediawiki/2013/f/ff/PrG_R.jpg"> <img src="https://static.igem.org/mediawiki/2013/f/ff/PrG_R.jpg" width="600">
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<p style="text-align:center; color:black; "> <b>Figure 15:</b>Alignment of the full tat_ProteinG construct. </p> </div>
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<p>The Protein G gene from the <i>Streptococcus dysgalactiae ssp equisimilis</i> sample we collected from the hospital is clearly missing two bigger segments compared to the <a href="http://www.ncbi.nlm.nih.gov/nuccore/X06173"> known gene sequence</a>. We conclude that this is Protein G as the rest of the sequence aligns well.<br>
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<p>To check weather Protein G is even produced in the S3-2B cells we did a SDS-PAGE with these cells along with a wildtype ER2566 cell sample (see figure below):<br><br>
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<div class="col4" style="background-color:white;><a href="https://static.igem.org/mediawiki/2013/1/14/Bakteri_SDS_gel_S3.jpg"> <img src="https://static.igem.org/mediawiki/2013/1/14/Bakteri_SDS_gel_S3.jpg" width="500">
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<p style="text-align:center; color:black; "><b> Figure 17:</b>SDS-PAGE of wildtype (WT) ER2566 cells and ER2566 S3-2B cells with tat_ProteinG construct. Ladder applied is Precision Plus Protein<sup>TM</sup> Unstained Standards.</p> </div>
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INCERT MARIAS STUFF ON VESICLES.
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There is a very clear additional band on the S3-2B sample of about 60 kDa in protein mass. Highly indicating that tat_Protein G is produced in the cells. <br>
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The <b>conclusion</b> is that Protein G is obviously produced in the <i>E.coli</i> strain ER2566 cells, but the tat signal peptide seems to fail to direct the Protein G into the periplasm and OMVs. <br>

Latest revision as of 21:30, 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). Sequencing confirms that we have succesfully made our tat_GFP_RFP construct.
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


Pm/xylS promoter



The Pm/xylS promoter was modified (removing an XbaI site) and turned into a BioBrick by adding the prefix and suffix. With 3A assembly, the promoter was attached to a GFP generator (BBa_E0240), and a backbone ( pSB3K3). The results show that the GFP are expressed in the samples with the Pm/xylS promoter, indicating that the promoter works. The figures below show the result from the testing. As expected, the reference promoter has a high rate of GFP synthesis.

Figure X: The ratio of GFP synthesis as a function of inducer concentration

Figure X: The ratio of GFP synthesis as a function of time



Protein G





We have introduced Protein G from Streptococcus dysgalactiae ssp equisimilis into E.coli where it is succesfully expressed.



Figure 15:Alignment of the full tat_ProteinG construct.



The Protein G gene from the Streptococcus dysgalactiae ssp equisimilis sample we collected from the hospital is clearly missing two bigger segments compared to the known gene sequence. We conclude that this is Protein G as the rest of the sequence aligns well.

To check weather Protein G is even produced in the S3-2B cells we did a SDS-PAGE with these cells along with a wildtype ER2566 cell sample (see figure below):

Figure 17:SDS-PAGE of wildtype (WT) ER2566 cells and ER2566 S3-2B cells with tat_ProteinG construct. Ladder applied is Precision Plus ProteinTM Unstained Standards.



There is a very clear additional band on the S3-2B sample of about 60 kDa in protein mass. Highly indicating that tat_Protein G is produced in the cells.
The conclusion is that Protein G is obviously produced in the E.coli strain ER2566 cells, but the tat signal peptide seems to fail to direct the Protein G into the periplasm and OMVs.

Retrieved from "http://2013.igem.org/Team:NTNU-Trondheim/Achievements"