Difference between revisions of "Team:Freiburg/Collaborations"

 
(35 intermediate revisions by 11 users not shown)
Line 2: Line 2:
 
{{Freiburg/Menubar}}
 
{{Freiburg/Menubar}}
  
{{Freiburg/wiki_content_start}}
+
{{Team:Freiburg/wiki_content_start_bubble}}
 
<html>
 
<html>
 
<style>
 
<style>
 
/*========= BEGIN: style for navigation bar ==========*/
 
/*========= BEGIN: style for navigation bar ==========*/
#team {
+
#results {
     background: url(https://static.igem.org/mediawiki/2015/a/a9/Freiburg_icon_team_active_yellow.png) no-repeat;
+
     background: url(https://static.igem.org/mediawiki/2015/2/2e/Freiburg_icon_results_active_yellow.png) no-repeat;
 
}
 
}
  
#team a {
+
#results a {
 
     color: #ecdc18;
 
     color: #ecdc18;
 
}
 
}
  
 
#runningchip {
 
#runningchip {
     left: 35.5%;
+
     left: 52%;
 
}
 
}
 
/*========= END: style for navigation bar ==========*/
 
/*========= END: style for navigation bar ==========*/
 
</style>
 
</style>
 +
<script type="text/javascript">
 +
//===================BEGIN:Amazing Bubble Sidebar==========================
  
<div class="kommentar">
+
$(document).ready(function(){
Kommen hier noch zumindest noch ein paar Daten rein?
+
  // CHANGE THE FOLLOWING ATTRIBUTES //
Warum ist Bifield in Blockquote und stockholm nicht?
+
  var href_text1='https://2015.igem.org/Team:Freiburg/Results',
</div>
+
  // Text2 needs no href as it is the actual page //
 +
  img_url='https://static.igem.org/mediawiki/2015/1/1e/Freiburg_icon_results_white_03.png',
 +
  href_text3='https://2015.igem.org/Team:Freiburg/InterLab_Study',
 +
  // Text1 needs no text as it is a pic //
 +
  text2='Collaborations';
 +
  // Text3 needs no text as its always 'next' //
 +
  // HOLD ON CHANGING THINGS --JABBERWOCK  //
 +
 
 +
  $('#bubble1').attr('href',href_text1);
 +
  $('#bubble1_img').attr('src', img_url);
 +
  $('#bubble3').attr('href',href_text3);
 +
 
 +
  $('#bubble2').text(text2);
 +
});
 +
 
 +
//===================END:Amazing Bubble Sidebar==========================
 +
</script>
 
<div class="content_box">
 
<div class="content_box">
 
<!-- Labjournal content goes in here -->
 
<!-- Labjournal content goes in here -->
 
  
 
<h1 class="sectionedit1">Collaboration with iGEM Team Bielefeld</h1>
 
<h1 class="sectionedit1">Collaboration with iGEM Team Bielefeld</h1>
 
<div class="level1">
 
<div class="level1">
 
<blockquote class="blockquote-plugin">
 
<blockquote class="blockquote-plugin">
<p>
 
  
<strong>Bielefeld</strong> sends a plasmid based on <a class="urlextern" href="http://parts.igem.org/Part:BBa_I746909" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_I746909">BBa_I746909</a> that has a translation enhancing sequence (5’-UTR), and <strong>Freiburg</strong> sends a plasmid containing turboYFP, a His- and a Halo-Tag. We would like to compare if these parts work in different cell-free proteins synthesis environments.
+
 
</p>
+
</blockquote>
+
 
<div class="image_box right">
 
<div class="image_box right">
<a class="media lightbox_trigger" href="https://static.igem.org/mediawiki/2015/f/f8/Freiburg_bielefeld_collab.jpg" title="bielefeld_collab.jpg"><img align="right" alt="" class="mediaright" src="https://static.igem.org/mediawiki/2015/f/f8/Freiburg_bielefeld_collab.jpg" width="200"/></a>
+
<a class="media lightbox_trigger" href="https://static.igem.org/mediawiki/2015/f/f8/Freiburg_bielefeld_collab.jpg" title="bielefeld_collab.jpg"><img align="right" alt="" class="mediaright" src="https://static.igem.org/mediawiki/2015/f/f8/Freiburg_bielefeld_collab.jpg" width="140"/></a>
 
</div>
 
</div>
 
<p>
 
<p>
To test the plasmid BBa_I746909 containing a translation enhancing sequence (5'-UTR) we compared it to our GFPs used for cell-free expression (HA-GFP-His6-His6 and His-GFP-Spy).
+
Team <strong>Bielefeld</strong> sent us  a plasmid based on <a class="urlextern" href="http://parts.igem.org/Part:BBa_I746909" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_I746909">BBa_I746909</a> having a translation enhancing sequence (5’-UTR). We, the iGEM team <strong>Freiburg</strong>, sent a plasmid containing coding sequences for turboYFP, a His- and a Halo-Tag. We aimed to analyze if these parts will work in different cell-free protein synthesis environments.
 +
</p>
 +
</blockquote>
  
Both plasmids were treated alike and compared to a sample containing no DNA (negative control) and a dilution series of expressed and purified GFP (positive control). All reactions were performed in triplicates.
+
<p>
 +
To test the plasmid <a class="urlextern" href="http://parts.igem.org/Part:BBa_I746909" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_I746909">BBa_I746909</a>  containing a translation enhancing sequence (5'-UTR) we performed a cell-free expression with our own mix and compared it to a sample containing no DNA (negative control). <br>
  
The samples were expressed for 2 hours at 37°C in a 384-well plate using our own lysate and premix. After expression, a western blot and dot blot were performed.
+
Both samples were treated alike and compared. All reactions were performed in duplicates.
 +
 
 +
The samples were expressed for 4 hours at 37°C in a 384-well plate using our DiaMIX. After expression, the graph clearly indicates successful expression of GFP. This underlines the ability of the DiaMIX to express vectors from various sources and the effectiveness of a 5'UTR.  
 
</p>
 
</p>
 +
 +
    <div class="image_box center">
 +
        <div class="thumb2 trien" style="width:600px">
 +
            <div class="thumbinner">
 +
                <a href="https://static.igem.org/mediawiki/2015/a/a2/Freiburg_bilefeld.png" class="lightbox_trigger">
 +
                    <img src="https://static.igem.org/mediawiki/2015/a/a2/Freiburg_bilefeld.png" width="400">
 +
                </a>
 +
                <div class="thumbcaption">
 +
                    <p>
 +
                        <strong>Figure 1: Expression of <a class="urlextern" href="http://parts.igem.org/Part:BBa_I746909" rel="nofollow" target="_Blank" title="http://parts.igem.org/Part:BBa_I746909">BBa_I746909.</a> </strong> The expression was conducted for 4 h and relative fluorescence measured in one minute steps. Mean values and standard deviations were calculated from duplicates.
 +
      </p>
 +
                </div>
 +
            </div>
 +
        </div>
 +
    </div>
 +
 +
 
</div>
 
</div>
 
</div>
 
</div>
Line 55: Line 91:
 
<blockquote class="blockquote-plugin">
 
<blockquote class="blockquote-plugin">
 
<p>
 
<p>
The iGEM Team Stockholm wants to develop a bacterial biomarker for early detection of cancer biomarkers. We thought as we are both working on diagnostic tools it would be great to start a collaboration.
+
<strong>Stockholm</strong> sent us the lysate of a HER2 domain binding affibody with a His-tag as well as a glycerol stock of the <i>E. coli</i> Top10 strain they used for expression and organized the sponsoring of the HER2-antigen from R&D-systems.  
 +
<br/><strong>Freiburg</strong> tried to measure the binding of the purified HER2-antigen to the His-affibody lysate in iRIf.
 
</p>
 
</p>
 
</blockquote>
 
</blockquote>
 +
 +
 +
    <div class="image_box right">
 +
        <div class="thumb2 trien" style="width:300px">
 +
            <div class="thumbinner">
 +
                <a href="https://static.igem.org/mediawiki/2015/f/f1/Freiburg_wb_membrane_stained_affibody.jpeg" class="lightbox_trigger">
 +
                    <img src="https://static.igem.org/mediawiki/2015/f/f1/Freiburg_wb_membrane_stained_affibody.jpeg" width="280">
 +
                </a>
 +
                <div class="thumbcaption">
 +
                    <p>
 +
                        <strong>Figure 1: Amido black stained Western Blot of His-tagged affibody.</strong> No signal could be detected at 10 kDa (size of the affibody)
 +
      </p>
 +
                </div>
 +
            </div>
 +
        </div>
 +
    </div>
  
 
<p>
 
<p>
Therefore, we wanted to measure with our DiaChip the interaction of one of their affibodies to the Her2-protein, one of their cancer biomarkers. Team Stockholm expressed the affibody with a His-Tag, so it would bind to our Ni-NTA surface and sent the "E. coli" lysate to us. From R&amp;D-Systems we reveived the purified Her2-antigen. Unfortunately, the antigen also had a His-Tag, so we couldn't measure it on Ni-NTA slides, because it would not only bind the affibody but also the Ni-NTA on the surface. This would lead to an increased optical thickness all over the slide and we would not be able to get a specific signal. If we had spotted the affibody lysate on an unspecific surface, all other proteins in the lysate would have bound to the surface as well. This would have resulted in an affibody concentration on the spot below the detection limit. So that wasn't a possibility either.
+
As the iGEM Team Stockholm 2015 also works on a diagnostic tool, we thought it would be great to combine our two approaches. For early detection of cancer biomarkers, Stockholm tried to establish an affibody based bacterial biomarker assay (ABBBA). We planed to measure the binding of the purified HER2-antigen to the corresponding His-tagged affibody Stockholm sent us, which we immobilized on our Ni-NTA surface. Unfortunately, we realized that the HER2-antigen was also His-tagged, so it was not suitable for an anlysis on a Ni-NTA surface. The HER2-antigen would bind to the surface, precluding a detection of its binding to the affibody.  
 
</p>
 
</p>
 
<p>
 
<p>
Our last chance was to spot the antigen on an unspecific surface and flush over the affibody lysate. Due to the small molecular weight of the affibody (10 kDa), it is really hard to see a binding in the iRIf (detection limit: ~10 kDa). Nevertheless we still tried.
+
To circumvent this problem we spotted the HER2-antigen on a PDITC surface and measured the binding of the affibody to the protein. Due to the small size of the affibody (under 10 kDa) it's not possible to observe the binding with iRIf because the detection limit of the system is around 10 kDa.
 +
</p>
 +
<p>
 +
A third possibility would have been to spot the His-tagged affibody on a PDITC surface and flush the slide with the antigen. Therefore, it would have been necessary to purify the affibody, so we expressed it freshly from the glycerol stock Stockholm sent us. We performed a Western Blot of the pellet and the soluble fraction after lysis of the <i>E.coli</i> cells that expressed the affibody. To identify the expressed protein we used Ni-NTA conjugated HRP antibody. Additionally, we stained the membrane with amidoblack to determine successful transfer of the proteins onto the membrane (figure 1). Unfortunately, there was no signal detectable by chemiluminescence at the expected molecular weight. Therefore, we did not proceed with purification.
 +
 
 +
 
 
</p>
 
</p>
 
<div class="tags"><span>
 
<div class="tags"><span>
Line 77: Line 135:
 
<blockquote class="blockquote-plugin">
 
<blockquote class="blockquote-plugin">
 
<p>
 
<p>
We contributed to the iGEM Newsletter published by the iGEM Team Amoy.
+
We contributed to the iGEM Newsletter published by the <strong>iGEM Team Amoy</strong>.
 
</p>
 
</p>
 
</blockquote>
 
</blockquote>
Line 86: Line 144:
  
 
<p>
 
<p>
The iGEM Team Amoy published regular Newsletters for the iGEM competition together with Paris_Bettencourt and Pasteur_Paris. It provided all iGEM Teams with the possibility to share their project idea, information about experiments they are performing or opinions about crucial topics of synthetic biology as well as to ask for help with complications they faced during summer. All in all, there were seven issues published with different contents, three of them being special issues dealing with setting up an iGEM Team, the current situation of synthetic biology and software used in iGEM.
+
The iGEM Team Amoy published a regular Newsletters for the iGEM competition together with Paris_Bettencourt and Pasteur_Paris. They provided all iGEM Teams with the possibility to share their project idea, information about experiments they are performing or opinions about critical topics of synthetic biology as well as to ask for help with complications they faced during this summer. All in all, there were seven issues published with different contents, three of them being special issues dealing with setting up an iGEM Team, the current situation of synthetic biology and software used in iGEM.
 
</p>
 
</p>
 
<p>
 
<p>
We were asked if we would like to contribute to the work of the iGEM Team and used this opportunity to share our project and thoughts with the iGEM community. We were pleased to be able to contribute to such a great piece of work.
+
We were asked to contribute to the work of the iGEM Team and used this opportunity to share our project and thoughts with the iGEM community. We were delighted to be able to contribute to such a great piece of work.
 
</p>
 
</p>
 
<p>
 
<p>
All the issues that were published during this year's iGEM competition and further information can be found <a class="urlextern" href="https://2015.igem.org/Team:Amoy/Newsletter#title" rel="nofollow" target="_Blank" title="https://2015.igem.org/Team:Amoy/Newsletter#title">here</a>.
+
All issues that were published during this year's iGEM competition and further information can be found <a class="urlextern" href="https://2015.igem.org/Team:Amoy/Newsletter#title" rel="nofollow" target="_Blank" title="https://2015.igem.org/Team:Amoy/Newsletter#title">here</a>.
 
</p>
 
</p>
 
</div>
 
</div>
Line 103: Line 161:
 
<blockquote class="blockquote-plugin">
 
<blockquote class="blockquote-plugin">
 
<p>
 
<p>
The iGEM Team Tübingen provided us with a Spy-tagged protein.
+
The <strong>iGEM Team Tübingen</strong> provided us with a construct coding for the Spy-Tag.
 
</p>
 
</p>
 
</blockquote>
 
</blockquote>
  
 
<div class="image_box right">
 
<div class="image_box right">
<a class="media" href="https://static.igem.org/mediawiki/2015/c/c4/Freiburg_Tuebingen_Collab.jpg" title="Freiburg_Tuebingen_Collab"><img align="right" alt="" class="mediaright" src="https://static.igem.org/mediawiki/2015/c/c4/Freiburg_Tuebingen_Collab.jpg" width="200"/></a>
+
<a class="media lightbox_trigger" href="https://static.igem.org/mediawiki/2015/c/c4/Freiburg_Tuebingen_Collab.jpg" title="Freiburg_Tuebingen_Collab"><img align="right" alt="" class="mediaright" src="https://static.igem.org/mediawiki/2015/c/c4/Freiburg_Tuebingen_Collab.jpg" width="200"/></a>
 
</div>
 
</div>
  
  
 
<p>
 
<p>
For the establishment of a specific surface (LINK ZU SurCHEM!!!!!) in the procedure of producing our DiaCHIP we followed different approaches. We planned to, among others, test a specific surface with the SpyCatcher on top and the expression of Spy-tagged proteins. In order to be able to test our surface before performing cell-free expression (LINK ZU CELL-FREE), the iGEM Team Tübingen kindly provided us with a purified antigen with a Spy tag fused to it.
+
For the establishment of a <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Results/Surface">specific surface</a> on the glass slide of the DiaCHIP we followed different approaches. Among others, we planned to test a surface with SpyCatcher immobilized on top of an activated silane layer. The SpyCatcher would then interact specifically with Spy-tagged proteins. In order to be able to verify the predicted function of our surface before performing <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Results/Cellfree">cell-free expression</a>, the iGEM Team Tübingen kindly provided us with a purified carboxyfluorescine labeled <a href="http://parts.igem.org/Part:BBa_K1159201"target="_blank">SpyTag</a>.  
 
</p>
 
</p>
 
<p>
 
<p>
Unfortunately, due to time constraints, we were not able to establish a SpyCatcher surface and test the protein we received.
+
Unfortunately, we were not able to establish a SpyCatcher surface and test the corresponding SpyTag we received from Team Tübingen due to time constraints.
 
</p>
 
</p>
 
</div>
 
</div>

Latest revision as of 02:36, 19 September 2015

""

Collaboration with iGEM Team Bielefeld

Team Bielefeld sent us a plasmid based on BBa_I746909 having a translation enhancing sequence (5’-UTR). We, the iGEM team Freiburg, sent a plasmid containing coding sequences for turboYFP, a His- and a Halo-Tag. We aimed to analyze if these parts will work in different cell-free protein synthesis environments.

To test the plasmid BBa_I746909 containing a translation enhancing sequence (5'-UTR) we performed a cell-free expression with our own mix and compared it to a sample containing no DNA (negative control).
Both samples were treated alike and compared. All reactions were performed in duplicates. The samples were expressed for 4 hours at 37°C in a 384-well plate using our DiaMIX. After expression, the graph clearly indicates successful expression of GFP. This underlines the ability of the DiaMIX to express vectors from various sources and the effectiveness of a 5'UTR.

Figure 1: Expression of BBa_I746909. The expression was conducted for 4 h and relative fluorescence measured in one minute steps. Mean values and standard deviations were calculated from duplicates.

Collaboration with iGEM Team Stockholm

Stockholm sent us the lysate of a HER2 domain binding affibody with a His-tag as well as a glycerol stock of the E. coli Top10 strain they used for expression and organized the sponsoring of the HER2-antigen from R&D-systems.
Freiburg tried to measure the binding of the purified HER2-antigen to the His-affibody lysate in iRIf.

Figure 1: Amido black stained Western Blot of His-tagged affibody. No signal could be detected at 10 kDa (size of the affibody)

As the iGEM Team Stockholm 2015 also works on a diagnostic tool, we thought it would be great to combine our two approaches. For early detection of cancer biomarkers, Stockholm tried to establish an affibody based bacterial biomarker assay (ABBBA). We planed to measure the binding of the purified HER2-antigen to the corresponding His-tagged affibody Stockholm sent us, which we immobilized on our Ni-NTA surface. Unfortunately, we realized that the HER2-antigen was also His-tagged, so it was not suitable for an anlysis on a Ni-NTA surface. The HER2-antigen would bind to the surface, precluding a detection of its binding to the affibody.

To circumvent this problem we spotted the HER2-antigen on a PDITC surface and measured the binding of the affibody to the protein. Due to the small size of the affibody (under 10 kDa) it's not possible to observe the binding with iRIf because the detection limit of the system is around 10 kDa.

A third possibility would have been to spot the His-tagged affibody on a PDITC surface and flush the slide with the antigen. Therefore, it would have been necessary to purify the affibody, so we expressed it freshly from the glycerol stock Stockholm sent us. We performed a Western Blot of the pellet and the soluble fraction after lysis of the E.coli cells that expressed the affibody. To identify the expressed protein we used Ni-NTA conjugated HRP antibody. Additionally, we stained the membrane with amidoblack to determine successful transfer of the proteins onto the membrane (figure 1). Unfortunately, there was no signal detectable by chemiluminescence at the expected molecular weight. Therefore, we did not proceed with purification.

Collaboration with iGEM Team Amoy

We contributed to the iGEM Newsletter published by the iGEM Team Amoy.

The iGEM Team Amoy published a regular Newsletters for the iGEM competition together with Paris_Bettencourt and Pasteur_Paris. They provided all iGEM Teams with the possibility to share their project idea, information about experiments they are performing or opinions about critical topics of synthetic biology as well as to ask for help with complications they faced during this summer. All in all, there were seven issues published with different contents, three of them being special issues dealing with setting up an iGEM Team, the current situation of synthetic biology and software used in iGEM.

We were asked to contribute to the work of the iGEM Team and used this opportunity to share our project and thoughts with the iGEM community. We were delighted to be able to contribute to such a great piece of work.

All issues that were published during this year's iGEM competition and further information can be found here.

Collaboration with iGEM Team Tübingen

The iGEM Team Tübingen provided us with a construct coding for the Spy-Tag.

For the establishment of a specific surface on the glass slide of the DiaCHIP we followed different approaches. Among others, we planned to test a surface with SpyCatcher immobilized on top of an activated silane layer. The SpyCatcher would then interact specifically with Spy-tagged proteins. In order to be able to verify the predicted function of our surface before performing cell-free expression, the iGEM Team Tübingen kindly provided us with a purified carboxyfluorescine labeled SpyTag.

Unfortunately, we were not able to establish a SpyCatcher surface and test the corresponding SpyTag we received from Team Tübingen due to time constraints.