Difference between revisions of "Team:Aalto-Helsinki/Parts"

m
(actually GFP was fused with amph template protein's PCR product, which is different from PCR product which was sent as a brick (we never used brick in the lab work), so changed brick to protein. (Clarification: only brick PCR product was sequenced))
 
(24 intermediate revisions by 3 users not shown)
Line 60: Line 60:
 
<h1>Submitted Parts</h1>
 
<h1>Submitted Parts</h1>
  
<section id="propane" class="active" data-anchor="propane">
+
<h2>Part Table </h2>
 +
</html>
 +
<groupparts>iGEM015 Aalto-Helsinki</groupparts>
 +
<html>
  
 +
<section id="propane" class="active" data-anchor="propane">
 +
<div id="propane_1"></div>
  
 
<h2>Propane 1 / BBa_K1655000</h2>
 
<h2>Propane 1 / BBa_K1655000</h2>
  
<figure style="float:right">
+
<figure style="float:right;margin-left:3%;">
 
   <img src="https://static.igem.org/mediawiki/2015/7/7c/Aalto-Helsinki_plasmid_propane1_for_white_bckgr.png" style="width:230px;"/>
 
   <img src="https://static.igem.org/mediawiki/2015/7/7c/Aalto-Helsinki_plasmid_propane1_for_white_bckgr.png" style="width:230px;"/>
 
   <figcaption><b><center>Figure 1.</b> Propane 1</center></figcaption>
 
   <figcaption><b><center>Figure 1.</b> Propane 1</center></figcaption>
 
</figure>
 
</figure>
  
<p>Our Propane 1 includes 4 of the 10 required genes to produce propane in <i>E. coli</i>. The plasmid has been assembled from IDT's gBlocks with NEBuilder assembly, similar to Gibson Assembly.</p>
+
<p>Our Propane 1 includes 3 of the 10 required genes to produce propane in <i>E. coli</i>. The plasmid has been assembled from IDT's gBlocks with NEBuilder assembly, similar to Gibson Assembly.</p>
<p><b>Validation:</b> We restricted our Propane 1 and ran the insert on an agarose gel. From the picture we can tell that the insert is the correct size. The result can be seen in <a href="https://static.igem.org/mediawiki/2015/3/3a/Aalto-Helsinki_car_submittedparts.png">Figure 2.</a><br/>
+
<p><b>Validation:</b> We restricted our Propane 1 and ran the insert on an agarose gel. From the picture we can tell that the plasmid's size is correct. The result can be seen in <a href="https://static.igem.org/mediawiki/2015/3/3a/Aalto-Helsinki_car_submittedparts.png">Figure 2.</a><br/>
 
Additionally, we did a colony PCR with VR and our primer P001. The VR primer attaches to our plasmid's backbone while P001 anneals with the very beginning of our construct. With this colony PCR we were able to show that the insert is present and it is indeed in the pSB1C3 backbone. See <a href="https://static.igem.org/mediawiki/2015/d/d1/Aalto-Helsinki_submittedparts_colony_pcr_validation.png">figure 3</a> for results, where the product in wells 1, and 5-10 is of the right size.</p>  
 
Additionally, we did a colony PCR with VR and our primer P001. The VR primer attaches to our plasmid's backbone while P001 anneals with the very beginning of our construct. With this colony PCR we were able to show that the insert is present and it is indeed in the pSB1C3 backbone. See <a href="https://static.igem.org/mediawiki/2015/d/d1/Aalto-Helsinki_submittedparts_colony_pcr_validation.png">figure 3</a> for results, where the product in wells 1, and 5-10 is of the right size.</p>  
 
<p>The colony which produced the product seen in figure 3, well 10 was sent to the registry under the name <b><a href="http://parts.igem.org/Part:BBa_K1655000">BBa_K1655000</a></b>.</p>
 
<p>The colony which produced the product seen in figure 3, well 10 was sent to the registry under the name <b><a href="http://parts.igem.org/Part:BBa_K1655000">BBa_K1655000</a></b>.</p>
Line 93: Line 98:
  
 
<section id="gfp" class="active" data-anchor="gfp">
 
<section id="gfp" class="active" data-anchor="gfp">
 +
<div id="gfp"></div>
 
<h2>Fusable GFP / BBa_K1655001</h2>
 
<h2>Fusable GFP / BBa_K1655001</h2>
  
<figure  style="float:right">
+
<figure  style="float:right;margin-left:3%;">
 
   <img src="https://static.igem.org/mediawiki/2015/1/1d/Aalto-Helsinki_gfp_plasmid_with_text.png" style="width:150px;"/>
 
   <img src="https://static.igem.org/mediawiki/2015/1/1d/Aalto-Helsinki_gfp_plasmid_with_text.png" style="width:150px;"/>
 
   <figcaption><center><b>Figure 4.</b> GFP Brick</center></figcaption>
 
   <figcaption><center><b>Figure 4.</b> GFP Brick</center></figcaption>
Line 101: Line 107:
  
 
<p>We built a GFP biobrick which can be fused to any protein's aminoterminal end with the standard BioBrick assembly enzymes. We have added an extra nucleotide prior to the brick's suffix to maintain the reading frame after fusion, which is typically lost when the restriction enzyme assembly is used. </p>
 
<p>We built a GFP biobrick which can be fused to any protein's aminoterminal end with the standard BioBrick assembly enzymes. We have added an extra nucleotide prior to the brick's suffix to maintain the reading frame after fusion, which is typically lost when the restriction enzyme assembly is used. </p>
<p><b>Validation:</b> Our GFP brick has been fully sequenced, and the sequencing results were as expected. See <span style="color:red">figure 5. </span>for the sequencing results. We have also been able to express the GFP after fusing it with an amphiphilic brick. This construct functioned under <a href="http://parts.igem.org/Part:BBa_K608003">BBa_K608003</a>, a strong constitutive promoter and a medium RBS. HS Slovenia Team also helped us validate this brick. They gained positive results of their construct with the GFP through colony PCR and analytical restrictions, but were unable to detect the fluerescence under UV light or functionality of the fused protein. We were however able to show that the GFP is functional after fusion. See <span style="color:red">figure 6.</span> for microscopic pictures.</p>
+
<p><b>Validation:</b> Our GFP brick has been fully sequenced, and the sequencing results were as expected. See figure 5. for the sequencing results. We have also been able to express the GFP after fusing it with an amphiphilic protein. This construct functioned under <a href="http://parts.igem.org/Part:BBa_K608003" target="_blank">BBa_K608003</a>, a strong constitutive promoter and a medium RBS. <a href="https://2015.igem.org/Team:Slovenia_HS">HS Slovenia Team</a> also helped us validate this brick. They gained positive results of their construct with the GFP through colony PCR and analytical restrictions, but were unable to detect the fluorescence under UV light or functionality of the fused protein. Figure 6 shows a positive result of colony PCR. The GFP has indeed been fused with another protein, CtfB, with the biobrick enzyme assembly.  We were however able to show that the GFP is functional after fusion. See figure 7. for microscopic pictures.</p>
<p style="color:red"> We restricted the GFP brick with XbaI & PstI to show that the insert in the brick was of the correct size. DNA from the colony which produced the band seen in Figure 7 in well 4 was sent to the registry under the name <b><a href="http://parts.igem.org/Part:BBa_K1655001">BBa_K1655001</a></b>.</p>
+
<p> We restricted the GFP brick with XbaI & PstI to show that the insert in the brick was of the correct size. DNA from the colony which produced the band seen in Figure 8 in well 4 was sent to the registry under the name <b><a href="http://parts.igem.org/Part:BBa_K1655001">BBa_K1655001</a></b>.</p>
 
<p>Click <a href="https://static.igem.org/mediawiki/2015/5/51/Aalto-Helsinki_gfp_sequence_ah009.gb">here</a> to download the full sequence of our Fusable GFP in pSB1C3 backbone.</p>
 
<p>Click <a href="https://static.igem.org/mediawiki/2015/5/51/Aalto-Helsinki_gfp_sequence_ah009.gb">here</a> to download the full sequence of our Fusable GFP in pSB1C3 backbone.</p>
  
 
<p>Click the images to enlarge them</p>
 
<p>Click the images to enlarge them</p>
 +
<div class="row">
 +
<div class="col-md-5">
 
<figure style="float:left">
 
<figure style="float:left">
   <a href="https://static.igem.org/mediawiki/2015/b/b3/Aalto-Helsinki_gfp_insert_validation_gel.png"><img src="https://static.igem.org/mediawiki/2015/b/b3/Aalto-Helsinki_gfp_insert_validation_gel.png" style="width:100px;"/></a>
+
   <a href="https://static.igem.org/mediawiki/parts/9/9e/Aalto-Helsinki_gfp_sequencing_results.png"><img src="https://static.igem.org/mediawiki/parts/9/9e/Aalto-Helsinki_gfp_sequencing_results.png" style="width:300px;"/></a>
   <figcaption><b><center><span style="color:red">Figure 5.</b> sequencing.</span></center></figcaption>
+
   <figcaption><b><center>Figure 5.</b> GFP brick's sequencing results</center></figcaption>
 
</figure>
 
</figure>
 +
</div>
 +
<div class="col-md-2">
 
<figure style="float:left">
 
<figure style="float:left">
   <a href="https://static.igem.org/mediawiki/2015/d/dc/Aalto-Helsinki_submittedparts_gfp_colonypcr_validation.png"><img src="https://static.igem.org/mediawiki/2015/d/dc/Aalto-Helsinki_submittedparts_gfp_colonypcr_validation.png" style="width:100px;"/></a>
+
   <a href="https://static.igem.org/mediawiki/2015/d/dc/Aalto-Helsinki_submittedparts_gfp_colonypcr_validation.png"><img src="https://static.igem.org/mediawiki/2015/d/dc/Aalto-Helsinki_submittedparts_gfp_colonypcr_validation.png" style="width:120px;"/></a>
   <figcaption><b><center>Figure 6.</b> Slovenia's colony PCR showing that GFP is fused with CtfB</center></figcaption>
+
   <figcaption><b>Figure 6.</b> Slovenia's colony PCR showing that GFP is fused with CtfB</figcaption>
 
</figure>
 
</figure>
 +
</div>
 +
<div class="col-md-2">
 
<figure style="float:left">
 
<figure style="float:left">
   <a href="https://static.igem.org/mediawiki/2015/b/b3/Aalto-Helsinki_gfp_insert_validation_gel.png"><img src="https://static.igem.org/mediawiki/2015/b/b3/Aalto-Helsinki_gfp_insert_validation_gel.png" style="width:102px;"/></a>
+
   <a href="https://static.igem.org/mediawiki/2015/9/99/Aalto-Helsinki_gfp_microscopic_images.png"><img src="https://static.igem.org/mediawiki/2015/9/99/Aalto-Helsinki_gfp_microscopic_images.png" style="width:90px;"/></a>
   <figcaption><b><center>Figure 7.</b> GFP brick restricted to show the right-sized insert.</center></figcaption>
+
   <figcaption><b>Figure 7.</b> Microscopic images of GFP fusion</center></figcaption>
 
</figure>
 
</figure>
 +
</div>
 +
<div class="col-md-2">
 +
<figure style="float:left;margin-bottom:2%;">
 +
  <a href="https://static.igem.org/mediawiki/2015/b/b3/Aalto-Helsinki_gfp_insert_validation_gel.png"><img src="https://static.igem.org/mediawiki/2015/b/b3/Aalto-Helsinki_gfp_insert_validation_gel.png" style="width:90px;"/></a>
 +
  <figcaption><b>Figure 8.</b> GFP brick restricted to show the right-sized insert.</center></figcaption>
 +
</figure>
 +
</div>
 +
</div>
 +
 +
<p>This brick is a twin to three different bricks. We checked the BioBrick seeker for any similar bricks before we started with our project, and the twin for this part did not come up. We only realized that a brick like this had already been created after we submitted our parts, which was about a week prior to the wiki freeze.</p>
  
 
<div class="row"></div>
 
<div class="row"></div>
Line 125: Line 147:
  
 
<section id="amph" class="active" data-anchor="amph">
 
<section id="amph" class="active" data-anchor="amph">
 +
<div id="amphiphilic"></div>
 
<h2>Amphiphilic / BBa_K1655002</h2>
 
<h2>Amphiphilic / BBa_K1655002</h2>
  
<figure  style="float:right">
+
<figure  style="float:right;margin-left:3%;">
  <img src="https://static.igem.org/mediawiki/2015/f/f7/Aalto-Helsinki_amph_plasmid_with_text.png" style="width:150px;"/>
+
<img src="https://static.igem.org/mediawiki/2015/f/f7/Aalto-Helsinki_amph_plasmid_with_text.png" style="width:150px;"/>
   <figcaption><center><b>Figure 5.</b> Amphiphilic Brick</center></figcaption>
+
   <figcaption><center><b>Figure 9.</b> Amphiphilic Brick</center></figcaption>
 
</figure>
 
</figure>
 +
<p>We submitted a sole amphiphilic brick, containing only the coding region of the ampihphilic protein. This brick can be used to produce intracellular micelles or vesicles under any chosen expression system. Our amphiphilic protein is constructed so that the hydrophilic part is translated first. According to <a href="http://www.nature.com/nmat/journal/v14/n1/abs/nmat4118.html">Huber <i>et al.</i></a> this allows for the formation on of intracellular micelles and vesicles. If the hydrophobic domain was to be translated first, the amphiphils would only form micelles.<p>
  
<p>We submitted a sole amphiphilic brick, containing only the coding region of the ampihphilic protein. This brick can be used to produce intracellular micelles or vesicles under any chosen expression system.</p>
+
<p><b>Validation:</b> Our amphiphilic brick's sequencing results were unclear, as the brick is mainly built of short repeats. We have however been able to show that the size on this insert in the pSB1C3 backbone is correct. See figure 10 for a gel electrophoresis image. The DNA from the colony which produced the product seen in well 2 was sent to the registry. We were also able to show, that once amphiphilic protein is fused to a GFP, the GFP is expressed and again, the construct size is correct. See figure 11 where the last two wells before the ladder show a correct plasmid size. Although the sequencing results are unclear, the correct sized mRNA seemed to be produced. This lead us to believe, that the Amphiphilic protein was indeed transcribed and possibly translated as well. We thought that we weren't able to detect the micelles and vesicles because of low accuracy of the microscopic images or because our expression level was too high. Our promoter was 10 times more efficient than the one that has been previously used to produce micelles and vesicles with this amphiphilic protein. The high concentration of amphiphils is likely to interfere with the micelle and vesicle formation.<br/><br/></p>
<p><b>Validation:</b> Our amphiphilic brick's sequencing results were unclear, as the brick is mainly built of short repeats. <span style="color:red">Microscope pictures!</span></p>
+
<p style="color:red">The colony which produced the product seen in figure 3, well 10 was sent to the registry under the name <b><a href="http://parts.igem.org/Part:BBa_K1655002">BBa_K1655002</a></b>.</p>
+
<p>Click <a href="https://static.igem.org/mediawiki/2015/4/41/Aalto-Helsinki_amph_sequence_ah009.gb">here</a> to download the full sequence of the Amphiphilic Brick in pSB1C3 backbone.</p>
+
  
 +
<p><span style="color:red"><b>Note!</b></span> The submitted BioBrick does most likely not contain the correct sequence. Restriction analysis lead us to initially believe we had the correct insert, as the insert size in pSB1C3 backbone matched that of the correct sequence (figure 10). However, as we proceeded to the sequencing results of the then already submitted brick a day before the wiki freeze, we found out that the sequence was not that of the amphiphilic protein, but instead that of the <a href="http://parts.igem.org/Part:BBa_K592009">blue chromoprotein</a>.</p>
  
 +
<p>Click the images to enlarge them</p>
 +
<div class="row">
 +
<div class="col-md-5">
 +
<figure  style="float:left">
 +
  <a href="https://static.igem.org/mediawiki/2015/c/c4/Aalto-Helsinki_amph_insert_validation_gel.png"><img src="https://static.igem.org/mediawiki/2015/c/c4/Aalto-Helsinki_amph_insert_validation_gel.png" style="width:100px;"/></a>
 +
  <figcaption><center><b>Figure 10.</b> Amphiphilic insert on gel</center></figcaption>
 +
</figure>
 +
</div>
 +
 +
<div class="col-md-2">
 +
<figure  style="float:left">
 +
  <a href="https://static.igem.org/mediawiki/2015/6/67/Aalto-Helsinki_amph_gfp_fusion_ongel.png"><img src="https://static.igem.org/mediawiki/2015/6/67/Aalto-Helsinki_amph_gfp_fusion_ongel.png" style="width:100px;"/></a>
 +
  <figcaption><center><b>Figure 11.</b> GFP-Amphiphilic fusion on gel</center></figcaption>
 +
</figure>
 +
</div>
 +
</div>
 
</section>
 
</section>
  
  
<h2> Part Documentation</h2>
 
 
<p>Each team will make new parts during iGEM and will submit them to the Registry of Standard Biological Parts. The iGEM software provides an easy way to present the parts your team has created. The <code>&lt;groupparts&gt;</code> tag (see below) will generate a table with all of the parts that your team adds to your team sandbox.</p>
 
<p>Remember that the goal of proper part documentation is to describe and define a part, so that it can be used without needing to refer to the primary literature. Registry users in future years should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.</p>
 
 
<h4>Part Table </h4>
 
</html>
 
<groupparts>iGEM015 Aalto-Helsinki</groupparts>
 
<html>
 
  
  
  
<p style="margin-bottom:0;padding-bottom:10%;"></p>
+
<p style="margin-bottom:0;padding-bottom:10%;clear: both;"></p>
  
 
</div>
 
</div>

Latest revision as of 04:59, 29 October 2015

Submitted Parts

Part Table

<groupparts>iGEM015 Aalto-Helsinki</groupparts>

Propane 1 / BBa_K1655000

Figure 1. Propane 1

Our Propane 1 includes 3 of the 10 required genes to produce propane in E. coli. The plasmid has been assembled from IDT's gBlocks with NEBuilder assembly, similar to Gibson Assembly.

Validation: We restricted our Propane 1 and ran the insert on an agarose gel. From the picture we can tell that the plasmid's size is correct. The result can be seen in Figure 2.
Additionally, we did a colony PCR with VR and our primer P001. The VR primer attaches to our plasmid's backbone while P001 anneals with the very beginning of our construct. With this colony PCR we were able to show that the insert is present and it is indeed in the pSB1C3 backbone. See figure 3 for results, where the product in wells 1, and 5-10 is of the right size.

The colony which produced the product seen in figure 3, well 10 was sent to the registry under the name BBa_K1655000.

Click here to download the full sequence of Propane 1 in pSB1C3 backbone.

Click the images to enlarge them.

Figure 2. Restriction digestion
Figure 3. Propane 1 colony PCR with primers P001 & VR

Fusable GFP / BBa_K1655001

Figure 4. GFP Brick

We built a GFP biobrick which can be fused to any protein's aminoterminal end with the standard BioBrick assembly enzymes. We have added an extra nucleotide prior to the brick's suffix to maintain the reading frame after fusion, which is typically lost when the restriction enzyme assembly is used.

Validation: Our GFP brick has been fully sequenced, and the sequencing results were as expected. See figure 5. for the sequencing results. We have also been able to express the GFP after fusing it with an amphiphilic protein. This construct functioned under BBa_K608003, a strong constitutive promoter and a medium RBS. HS Slovenia Team also helped us validate this brick. They gained positive results of their construct with the GFP through colony PCR and analytical restrictions, but were unable to detect the fluorescence under UV light or functionality of the fused protein. Figure 6 shows a positive result of colony PCR. The GFP has indeed been fused with another protein, CtfB, with the biobrick enzyme assembly. We were however able to show that the GFP is functional after fusion. See figure 7. for microscopic pictures.

We restricted the GFP brick with XbaI & PstI to show that the insert in the brick was of the correct size. DNA from the colony which produced the band seen in Figure 8 in well 4 was sent to the registry under the name BBa_K1655001.

Click here to download the full sequence of our Fusable GFP in pSB1C3 backbone.

Click the images to enlarge them

Figure 5. GFP brick's sequencing results
Figure 6. Slovenia's colony PCR showing that GFP is fused with CtfB
Figure 7. Microscopic images of GFP fusion
Figure 8. GFP brick restricted to show the right-sized insert.

This brick is a twin to three different bricks. We checked the BioBrick seeker for any similar bricks before we started with our project, and the twin for this part did not come up. We only realized that a brick like this had already been created after we submitted our parts, which was about a week prior to the wiki freeze.

Amphiphilic / BBa_K1655002

Figure 9. Amphiphilic Brick

We submitted a sole amphiphilic brick, containing only the coding region of the ampihphilic protein. This brick can be used to produce intracellular micelles or vesicles under any chosen expression system. Our amphiphilic protein is constructed so that the hydrophilic part is translated first. According to Huber et al. this allows for the formation on of intracellular micelles and vesicles. If the hydrophobic domain was to be translated first, the amphiphils would only form micelles.

Validation: Our amphiphilic brick's sequencing results were unclear, as the brick is mainly built of short repeats. We have however been able to show that the size on this insert in the pSB1C3 backbone is correct. See figure 10 for a gel electrophoresis image. The DNA from the colony which produced the product seen in well 2 was sent to the registry. We were also able to show, that once amphiphilic protein is fused to a GFP, the GFP is expressed and again, the construct size is correct. See figure 11 where the last two wells before the ladder show a correct plasmid size. Although the sequencing results are unclear, the correct sized mRNA seemed to be produced. This lead us to believe, that the Amphiphilic protein was indeed transcribed and possibly translated as well. We thought that we weren't able to detect the micelles and vesicles because of low accuracy of the microscopic images or because our expression level was too high. Our promoter was 10 times more efficient than the one that has been previously used to produce micelles and vesicles with this amphiphilic protein. The high concentration of amphiphils is likely to interfere with the micelle and vesicle formation.

Note! The submitted BioBrick does most likely not contain the correct sequence. Restriction analysis lead us to initially believe we had the correct insert, as the insert size in pSB1C3 backbone matched that of the correct sequence (figure 10). However, as we proceeded to the sequencing results of the then already submitted brick a day before the wiki freeze, we found out that the sequence was not that of the amphiphilic protein, but instead that of the blue chromoprotein.

Click the images to enlarge them

Figure 10. Amphiphilic insert on gel
Figure 11. GFP-Amphiphilic fusion on gel