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

m (picture fixes)
(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))
 
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<h1>Submitted Parts</h1>
 
<h1>Submitted Parts</h1>
 +
 +
<h2>Part Table </h2>
 +
</html>
 +
<groupparts>iGEM015 Aalto-Helsinki</groupparts>
 +
<html>
  
 
<section id="propane" class="active" data-anchor="propane">
 
<section id="propane" class="active" data-anchor="propane">
<div class="row">
+
<div id="propane_1"></div>
  
<div class="col-xs-12 col-sm-6">
+
<h2>Propane 1 / BBa_K1655000</h2>
<h2>Propane 1</h2>
+
  
<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>
+
<figure style="float:right;margin-left:3%;">
<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. <span style="color:red">Onko tähän jotain muuta? Sekvenssidataa tms?</span></p>  
+
  <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>
 +
</figure>
  
</div>
+
<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 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>
 +
<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>Click <a href="https://static.igem.org/mediawiki/2015/9/93/Aalto-Helsinki_car_sequence.gb">here</a> to download the full sequence of Propane 1 in pSB1C3 backbone.</p>
  
<div class="col-xs-12 col-sm-6">
+
<p>Click the images to enlarge them.</p>
 
+
<figure style="float:left; margin-right:2%;">
<figure style="margin-bottom:2%;margin-top:-4%;">
+
   <a href="https://static.igem.org/mediawiki/2015/3/3a/Aalto-Helsinki_car_submittedparts.png"><img src="https://static.igem.org/mediawiki/2015/3/3a/Aalto-Helsinki_car_submittedparts.png" style="width:150px;"/></a>
   <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 2.</b> Restriction digestion</center></figcaption>
   <figcaption><b>Figure 1.</b> Propane 1</figcaption>
+
 
</figure>
 
</figure>
  
  
<figure >
+
<figure style="float:left">
   <img src="https://static.igem.org/mediawiki/2015/3/3a/Aalto-Helsinki_car_submittedparts.png" style="width:280px;"/>
+
   <a href="https://static.igem.org/mediawiki/2015/d/d1/Aalto-Helsinki_submittedparts_colony_pcr_validation.png"><img src="https://static.igem.org/mediawiki/2015/d/d1/Aalto-Helsinki_submittedparts_colony_pcr_validation.png" style="width:102px;"/></a>
   <figcaption><b>Figure 2.</b> Propane 1 insert on gel</figcaption>
+
   <figcaption><b><center>Figure 3.</b> Propane 1 colony PCR with primers P001 & VR</center></figcaption>
 
</figure>
 
</figure>
</div>
 
  
</div><!-- row -->
+
<div class="row"></div>
  
 
</section>
 
</section>
  
 
<section id="gfp" class="active" data-anchor="gfp">
 
<section id="gfp" class="active" data-anchor="gfp">
<h2>Fusable GFP</h2>
+
<div id="gfp"></div>
 +
<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><b>Figure 3.</b> GFP Brick</figcaption>
+
   <figcaption><center><b>Figure 4.</b> GFP Brick</center></figcaption>
 
</figure>
 
</figure>
  
 
<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. We have also been able to express the GFP after fusing it with an amphiphilic brick. This construct functioned under 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 fluerescence under UV light or functionality of the fused protein.</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> 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>
  
</section>
+
<p>Click the images to enlarge them</p>
 
+
<div class="row">
 
+
<div class="col-md-5">
<section id="amph" class="active" data-anchor="amph">
+
<figure style="float:left">
<h2>Amphiphilic</h2>
+
  <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>Figure 5.</b> GFP brick's sequencing results</center></figcaption>
<figure style="float:right">
+
</figure>
   <img src="https://static.igem.org/mediawiki/2015/f/f7/Aalto-Helsinki_amph_plasmid_with_text.png" style="width:150px;"/>
+
</div>
   <figcaption><b>Figure 4.</b> Amphiphilic Brick</figcaption>
+
<div class="col-md-2">
 +
<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:120px;"/></a>
 +
   <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">
 +
  <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>Figure 7.</b> Microscopic images of GFP fusion</center></figcaption>
 +
</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>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>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>
<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>
+
  
 +
<div class="row"></div>
  
 
</section>
 
</section>
  
  
<h2> Part Documentation</h2>
+
<section id="amph" class="active" data-anchor="amph">
 +
<div id="amphiphilic"></div>
 +
<h2>Amphiphilic / BBa_K1655002</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>
+
<figure  style="float:right;margin-left:3%;">
<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>
+
<img src="https://static.igem.org/mediawiki/2015/f/f7/Aalto-Helsinki_amph_plasmid_with_text.png" style="width:150px;"/>
 +
  <figcaption><center><b>Figure 9.</b> Amphiphilic Brick</center></figcaption>
 +
</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><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>
  
<div class="highlightBox">
+
<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>
<h4>Note</h4>
+
 
<p>Note that parts must be documented on the <a href="http://parts.igem.org/Main_Page"> Registry</a>. This page serves to <i>showcase</i> the parts you have made. Future teams and other users and are much more likely to find parts by looking in the Registry than by looking at your team wiki.</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>
  
 +
<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>
  
  
<h4>Adding parts to the registry</h4>
 
<p>You can add parts to the Registry at our <a href="http://parts.igem.org/Add_a_Part_to_the_Registry">Add a Part to the Registry</a> link.</p>
 
<p>We encourage teams to start completing documentation for their parts on the Registry as soon as you have it available. The sooner you put up your parts, the better you will remember all the details about your parts. Remember, you don't need to send us the DNA sample before you create an entry for a part on the Registry. (However, you <b>do</b> need to send us the DNA sample before the Jamboree. If you don't send us a DNA sample of a part, that part will not be eligible for awards and medal criteria.)</p>
 
 
 
<h4>What information do I need to start putting my parts on the Registry?</h4>
 
<p>The information needed to initially create a part on the Registry is:</p>
 
<ul>
 
<li>Part Name</li>
 
<li>Part type</li>
 
<li>Creator</li>
 
<li>Sequence</li>
 
<li>Short Description (60 characters on what the DNA does)</li>
 
<li>Long Description (Longer description of what the DNA does)</li>
 
<li>Design considerations</li>
 
</ul>
 
 
<p>
 
We encourage you to put up <em>much more</em> information as you gather it over the summer. If you have images, plots, characterization data and other information, please also put it up on the part page. </p>
 
 
 
 
 
 
 
<h4>Inspiration</h4>
 
<p>We have a created  a <a href="http://parts.igem.org/Well_Documented_Parts">collection of well documented parts</a> that can help you get started.</p>
 
 
<p> You can also take a look at how other teams have documented their parts in their wiki:</p>
 
<ul>
 
<li><a href="https://2014.igem.org/Team:MIT/Parts"> 2014 MIT </a></li>
 
<li><a href="https://2014.igem.org/Team:Heidelberg/Parts"> 2014 Heidelberg</a></li>
 
<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Parts">2014 Tokyo Tech</a></li>
 
</ul>
 
 
 
 
<h4>Part Table </h4>
 
</html>
 
<groupparts>iGEM015 Example</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