Difference between revisions of "Team:Technion Israel/Collaborations"

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+
.intein_wrapper {
 
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//Code: inlarge img on click:
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$('.pop').on('click', function() {
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$('#imagemodal').modal('show'); 
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///pop-ups:
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$(document).ready(function(){
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<div class="intein_wrapper">
  
  
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<a href="#Peshawar" aria-controls="Peshawar" role="tab" data-toggle="tab">
 
<a href="#Peshawar" aria-controls="Peshawar" role="tab" data-toggle="tab">
 
<img src="https://static.igem.org/mediawiki/2016/b/b2/T--Technion_Israel--peshawar350x250.jpg" class="img-responsive img-center cont_tabs" width="175" height="125">
 
<img src="https://static.igem.org/mediawiki/2016/b/b2/T--Technion_Israel--peshawar350x250.jpg" class="img-responsive img-center cont_tabs" width="175" height="125">
−
<br><h4 class="text-center"><b>iGEM Peshawar</b></h4>
+
<br><h4 class="text-center"><b>Peshawar</b></h4>
 
</a>
 
</a>
 
</li>
 
</li>
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<a href="#Aachen" aria-controls="Aachen" role="tab" data-toggle="tab">
 
<a href="#Aachen" aria-controls="Aachen" role="tab" data-toggle="tab">
 
<img src="https://static.igem.org/mediawiki/2016/8/8f/T--Technion_Israel--Aachen350x250.jpg" class="img-responsive img-center cont_tabs" width="175" height="125">
 
<img src="https://static.igem.org/mediawiki/2016/8/8f/T--Technion_Israel--Aachen350x250.jpg" class="img-responsive img-center cont_tabs" width="175" height="125">
−
<br><h4 class="text-center"><b>iGEM Aachen</b></h4>
+
<br><h4 class="text-center"><b>Aachen</b></h4>
 
</a>
 
</a>
 
</li>
 
</li>
  
 
<li role="presentation" class="col-sm-3 col-xs-6">
 
<li role="presentation" class="col-sm-3 col-xs-6">
−
<a href="#BGU" aria-controls="BGU" role="tab" data-toggle="tab">
+
<a href="#Results" aria-controls="Results" role="tab" data-toggle="tab">
−
<img src="https://static.igem.org/mediawiki/2016/2/20/T--Technion_Israel--BGU350x250.jpg" class="img-responsive img-center cont_tabs" width="175" height="125">
+
<img src="https://static.igem.org/mediawiki/2016/c/c0/T--Technion_Israel--Eindhoven350x250.jpg" class="img-responsive img-center cont_tabs" width="175" height="125">
−
<br><h4 class="text-center"><b>iGEM BGU</b></h4>
+
<br><h4 class="text-center"><b>TU Eindhoven</b></h4>
 
</a>
 
</a>
 
</li>
 
</li>
  
 
<li role="presentation" class="col-sm-3 col-xs-6">
 
<li role="presentation" class="col-sm-3 col-xs-6">
−
<a href="#Eindhoven" aria-controls="Eindhoven" role="tab" data-toggle="tab">
+
<a href="#outlook" aria-controls="outlook" role="tab" data-toggle="tab">
−
<img src="https://static.igem.org/mediawiki/2016/c/c0/T--Technion_Israel--Eindhoven350x250.jpg" class="img-responsive img-center cont_tabs" width="175" height="125">
+
<img src="https://static.igem.org/mediawiki/2016/2/20/T--Technion_Israel--BGU350x250.jpg" class="img-responsive img-center cont_tabs" width="175" height="125">
−
<br><h4 class="text-center"><b>iGEM Eindhoven</b></h4>
+
<br><h4 class="text-center"><b>BGU</b></h4>
 
</a>
 
</a>
 
</li>
 
</li>
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</div>
 
</div>
 
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 +
 +
 +
 +
  
  
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<div class="tab-content">
 
<div class="tab-content">
  
−
<!-- =========== Peshawar =========== -->
+
 
−
<div role="tabpanel" class="tab-pane fade inactive" id="Peshawar">
+
 
 +
 
 +
<!-- =========== 1. Peshawar =========== -->
 +
<div role="tabpanel" class="tab-pane fade in active" id="Peshawar">
 
<div class="row"> <!--Headline-->
 
<div class="row"> <!--Headline-->
 
<div class="col-sm-12">
 
<div class="col-sm-12">
 
<br>
 
<br>
−
<h1 class="text-center"><u>iGEM - Peshawar</u></h1>
+
<h1 class="text-center"><u>Peshawar – Tutoring the first Pakistani team</u></h1>
 
</div>
 
</div>
 
</div>
 
</div>
−
 
  
 
<!-- ========== Content ========== -->
 
<!-- ========== Content ========== -->
−
 
−
 
  
 
<div class="row"><!-- #1 row -->
 
<div class="row"><!-- #1 row -->
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<div class="cont_box">
 
<div class="cont_box">
 
<div class="row">
 
<div class="row">
−
<a id="guidance"></a>
 
 
<div class="col-sm-12">
 
<div class="col-sm-12">
 
 
−
<div class="col-md-6 col-sm-12">
+
<div class="col-md-12 col-sm-12">
−
<h2 class="">Guidance</h2>
+
<h2 class="">Introduction</h2>
 
<p class="text-justify">
 
<p class="text-justify">
−
At the beginning we contacted Peshawar iGEM group because we discovered that both projects deal with detection.  
+
<b>We provided guidance to iGEM Peshawar, the first team to represent Pakistan. We assisted them with  
−
When we realized that the Peshawar group is the first team to represent Pakistan in iGEM we decided to share with  
+
various aspects of Mathematical Modelling, Human Practice and cloning.</b><br>
−
them the knowledge that was accumulated from past Technion iGEM teams. The first step was to open a joint Facebook
+
<br>
−
group, so each team member from every group would be able to find his colleague on the opposite team.
+
An opportunity for collaboration unveiled itself when we learned that the Pakistani team from Peshawar  
−
Later, we had Skype sessions regarding mathematical modeling, Human practices and Cloning.
+
University is focusing on detection as a project. During the initial conversation we were amazed to learn
 +
that said group is the first Pakistani team to compete in iGEM, thus they were in need of guidance with  
 +
most aspects of the competition. We decided to aid them and share all the knowledge and experience that  
 +
was accumulated in our project and in past Technion iGEM teams. To achieve this, we used different
 +
platforms for communications mainly a Facebook group, where the Peshawar team could get an immediate
 +
answer to any question they had, and multiple Skype sessions each with a different focus such as
 +
mathematical modeling, Human practices, Cloning and more.
 
</p>
 
</p>
 
</div>
 
</div>
−
 
−
<div class="col-md-6 col-sm-12 no-title-col">
 
−
<img src="http://freshdirtdesigns.com/blog/wp-content/uploads/2011/12/image_350x200_placeholder.png" class="img-responsive img-center img-cont">
 
−
</div>
 
−
 
 
</div>
 
</div>
 
</div>
 
</div>
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<div class="cont_box">
 
<div class="cont_box">
 
<div class="row">
 
<div class="row">
−
<a id="modeling"></a>
 
 
<div class="col-sm-12">
 
<div class="col-sm-12">
  
−
<div class="col-md-6 col-sm-12 new-row no-title-col">
+
<div class="col-md-12 col-sm-12">
−
<img src="http://freshdirtdesigns.com/blog/wp-content/uploads/2011/12/image_350x200_placeholder.png" class="img-responsive img-center img-cont">
+
−
</div>
+
−
 
+
−
<div class="col-md-6 col-sm-12">
+
 
<h2 class="">Mathematical modeling</h2>
 
<h2 class="">Mathematical modeling</h2>
 
<p class="text-justify">
 
<p class="text-justify">
−
We modeled Peshawar iGEM group's system using differential equations and logic gates. The aim of the work was  
+
We aided in the modeling process of Peshawar’s system, specifically using differential equations and logic gates.  
−
to predict the expression of the reporter protein when the concentration of the inducer is given.  
+
The aim of the model was to predict the expression of the reporter protein as a function of the inducers’ concentration.<br>
−
<b>צריך לבקש מנופר שתעבור על זה</b>  
+
</p>
 +
<p class="text-center">
 +
<a href="https://static.igem.org/mediawiki/2016/5/55/T--Technion_Israel--Peshawar_model.pdf" class="btn btn-primary"" target="_blank">View PDF<br></a>      
 
</p>
 
</p>
 
</div>
 
</div>
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<br>
 
<br>
 +
  
 
<div class="row"><!-- #3 row -->
 
<div class="row"><!-- #3 row -->
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<div class="cont_box">
 
<div class="cont_box">
 
<div class="row">
 
<div class="row">
−
<a id="human"></a>
 
 
<div class="col-sm-12">
 
<div class="col-sm-12">
  
 
<div class="col-md-6 col-sm-12">
 
<div class="col-md-6 col-sm-12">
−
<h2 class="">Human prctices</h2>
+
<h2 class="">Cloning:</h2>
 
<p class="text-justify">
 
<p class="text-justify">
−
We shared with Peshawar iGEM group the layout of our educational program.  
+
The Peshawar team had issues with the plasmid transformations into competent cells. After we got
−
In addition, we gave the team advices regarding an appeal to the government with a
+
a full report on the exact steps they followed, we helped debugging their protocol.<br>
−
request to promote the education in the field of Synthetic Biology.
+
We also shared our protocols of chemical transformation and competent cells preparation with them.<br>
 +
These instructions helped the team overcome their obstacles and proceed with the project.
 
</p>
 
</p>
 
</div>
 
</div>
  
−
<div class="col-md-6 col-sm-12 no-title-col">
+
 
−
<img src="http://freshdirtdesigns.com/blog/wp-content/uploads/2011/12/image_350x200_placeholder.png" class="img-responsive img-center img-cont">
+
<div class="col-md-6 col-sm-12">
 +
<h2 class="">Human practices:</h2>
 +
<p class="text-justify">
 +
We shared the layout of our educational program with Peshawar. Moreover, we advised the team regarding
 +
an appeal to the government with a request to promote the education in the field of Synthetic Biology.
 +
</p>
 
</div>
 
</div>
  
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<br>
 
<br>
 +
</div>
 +
<!-- =========== END: Intro =========== -->
  
−
<div class="row"><!-- #4 row -->
+
 
 +
 
 +
<!-- =========== 2: conection to the project =========== -->
 +
<div role="tabpanel" class="tab-pane fade" id="Aachen">
 +
<div class="row"> <!--Headline-->
 +
<div class="col-sm-12">
 +
<br>
 +
<h1 class="text-center"><u>Aachen- Prediction of mutated Subtilisin E protease</u></h1>
 +
</div>
 +
</div>
 +
 
 +
 
 +
<!-- ========== Content ========== -->
 +
 
 +
 
 +
<div class="row"><!-- #1 row -->
 
<div class="col-sm-10 col-sm-offset-1">
 
<div class="col-sm-10 col-sm-offset-1">
 
<div class="cont_box">
 
<div class="cont_box">
 
<div class="row">
 
<div class="row">
−
<a id="cloning"></a>
+
<div class="col-md-12 col-sm-12">
−
<div class="col-sm-12">
+
<h2 class="">Introduction</h2>
 +
<p class="text-justify">
 +
<b>We met with experts from the Technion to help us visualize the Subtilisin E and the photocaged serine
 +
in order to obtain insights regarding the structure of the mutated protein.</b><br>
 +
<br>
 +
We contacted iGEM Aachen when we learned their project deals with protein inactivation, as we believed it
 +
might correspond with our Intein sub-project. After discussing the details we came to the conclusion that
 +
the Intein protein would not be applicable to Aachen's system. Nevertheless, we decided to collaborate on
 +
different aspects of our projects.<br>
 +
<br>
 +
iGEM Aachen asked us to visualize and model the structure of the Subtilisin E protease, which is mutated
 +
with a photocaged serine as part of their project.<br>
 +
To do so we met with Prof. Meytal Landau (see: <a href="https://2016.igem.org/Team:Technion_Israel/Attribution">Attribution</a>)
 +
from the faculty of Biology at the Technion who provided information regarding different visualization tools available to aid us in this.<br>
 +
As we tried to simulate the photocaged serine ourselves using the Chemdraw software we encountered a few
 +
obstacles. In order to overcome these obstacles, we contacted Einav Tayab-Fligelman, from Prof. Landau's
 +
lab, whom kindly provided us with a PDB file with a visualization of the Photocaged serine.<br>
 +
Furthermore, we contacted Dr. Fabian Glaser (see: <a href="https://2016.igem.org/Team:Technion_Israel/Attribution">Attribution</a>)
 +
seeking assistance with the structure prediction. Dr. Glaser informed us that it would be impossible to
 +
receive a credible result within the short timeframe available. As an alternative he suggested writing
 +
a document describing the steps for an exhaustive computational work, including insights from the comparison
 +
of the structures of Subtilisin E and the photocaged serine.<br>
 +
Finally we created a 3D visualization of Subtilisin E adjacent to the photocaged serine along with the suggested document
 +
Dr. Glaser was kind enough to proofread and give his scientific remarks.
 +
</p>
  
−
<div class="col-md-6 col-sm12 new-row no-title-col">
+
<p class="text-center">
−
<img src="http://freshdirtdesigns.com/blog/wp-content/uploads/2011/12/image_350x200_placeholder.png" class="img-responsive img-center img-cont">
+
<a href="https://static.igem.org/mediawiki/2016/d/d1/T--Technion_Israel--Aachen_model.pdf" class="btn btn-primary"" target="_blank">View PDF<br></a>       
 +
</p>   
 +
 
 +
<p class="text-justify">
 +
The inevitable conclusion of the document was that the mutated Subtilisin E would not fold to the right tertiary
 +
structure. This information was highly valuable and helpful to Aachen’s project.<br>
 +
 
 +
<br>
 +
<br>
 +
 
 +
In return, we asked Aachen to build a biobrick consisting of the Tar chemoreceptor fused to a GFP marker.
 +
We provided them all the information necessary for this task.
 +
</p>
 +
<p class="text-center">
 +
<a href="https://static.igem.org/mediawiki/2016/e/e1/T--Technion_Israel--Aachen_TAR_GFP.pdf" class="btn btn-primary"" target="_blank">View PDF<br></a>       
 +
</p> 
 +
<p class="text-justify">
 +
One of the greatest challenges in forming a fusion is finding the right linker which is meant to bridge between
 +
the proteins and assure that the proteins fold in a correct manner. Since we had some trouble finding the right
 +
linker, the Aachen team suggested to fuse the Tar to the GFP without a linker. The Tar (K777000) and the GFP
 +
(E0040) biobricks were obtained from the iGEM kit. The expression system constructed by the Aachen iGEM team,
 +
also consisted of an expression backbone- Promoter and RBS (J04500) and a terminator (B0015).
 +
</p>
 +
</div>
 +
<div class="col-md-12 col-sm-12 new-row no-title-col">
 +
<a class="pop">
 +
<img src="https://static.igem.org/mediawiki/2016/5/57/T--Technion_Israel--AachenFig1.jpg" class="img-responsive img-center img-cont" style="cursor: pointer;"><br>
 +
</a>
 +
<p class="text-center"><b>Fig. 1:</b> The expression system which was constructed by the Aachen iGEM team,
 +
containing the Tar-GFP fusion. The current expression system does not include a linker.</p>
 
</div>
 
</div>
  
−
<div class="col-md-6 col-sm-12">
+
<div class="col-md-12 col-sm-12 new-row no-title-col">
−
<h2 class="">Cloning</h2>
+
 
<p class="text-justify">
 
<p class="text-justify">
−
Peshawar had issues with the transformation of plasmids into competent cells.
+
After building the expression system with the Tar receptor fused to GFP, the plasmid was transformed
−
They elaborated on the exact steps they did in the lab and we tried to debug their procedure.<br>
+
to a BL21 E. coli strain. To validate the expression, the cloned bacteria were tested in a Tecan plate
−
We also shared with team Peshawar our protocol for chemical transformation(3) and the protocol for producing competent cells(4). <br>
+
reader and under a fluorescent microscope.<br>
−
After following our instructions the Peshawar team managed to perform a successful transformation.
+
<br>
 +
GFP signal was detected on the polar part of the membrane only in a small fraction of cells. The majority
 +
of bacteria showed fluorescence in the entire volume of the cell, an indication of the accumulation of the
 +
Tar receptor inside the cell, probably due to an impaired structure of the protein (Fig. 2).
 
</p>
 
</p>
 
</div>
 
</div>
  
 +
<div class="col-md-6 col-sm-12 new-row no-title-col">
 +
<a class="pop">
 +
<img src="https://static.igem.org/mediawiki/2016/0/08/T--Technion_Israel--AachenFig2a.PNG" class="img-responsive img-center img-cont" width="350" height="350" style="cursor: pointer;"><br>
 +
</a>
 +
</div>
 +
 +
<div class="col-md-6 col-sm-12 new-row no-title-col">
 +
<a class="pop">
 +
<img src="https://static.igem.org/mediawiki/2016/6/67/T--Technion_Israel--AachenFig2b.PNG" class="img-responsive img-center img-cont" width="350" height="350" style="cursor: pointer;"><br>
 +
</a>
 +
</div>
 +
<div class="col-md-12 col-sm-12">
 +
<p class="text-center"><b>Fig. 2:</b> Fluorescent microscope results.</p>
 +
</div>
 +
 +
<div class="col-md-12 col-sm-12 new-row no-title-col">
 +
<p class="text-justify">
 +
The work made by Aachen has a major significance to our project and it gives decisive evidence to the
 +
importance of integrating a linker when fusing the proteins together. We would like to express our
 +
gratitude to the Aachen iGEM team for the valuable scientific work they have done for us.
 +
  </p>
 +
</div>
 +
</div>
 +
</div>
 +
</div>
 +
</div><!-- END: #1 row -->
 +
</div>
 +
<!-- =========== END: 2 - conection to the project =========== -->
 +
 +
 +
 +
 +
 +
 +
<!-- ==================== 3: TU Eindhoven ==================== -->
 +
<div role="tabpanel" class="tab-pane fade" id="Results">
 +
<div class="row"> <!--Headline-->
 +
<div class="col-sm-12">
 +
<br>
 +
<h1 class="text-center"><u>TU Eindhoven - Writing a manual for the Rosetta software</u></h1>
 +
</div>
 +
</div>
 +
 +
<!-- ========== Content ========== -->
 +
 +
<div class="row"><!-- #1 row -->
 +
<div class="col-sm-10 col-sm-offset-1">
 +
<div class="cont_box">
 +
<div class="row">
 +
<div class="col-sm-12">
 +
 +
<div class="col-md-12 col-sm-12">
 +
<h2 class="">Introduction</h2>
 +
<p class="text-justify">
 +
<b>We have written a manual for the operation of the Rosetta software together with iGEM TU Eindhoven.</b><br>
 +
<br>
 +
Our collaboration with iGEM Eindhoven was a result from the challenges we faced using the Rosetta software
 +
suite in our project. When we took our very first steps with protein modeling using Rosetta we quickly
 +
discovered numerous problems and difficulties that occupied us for weeks before we even started using the software.<br>
 +
During our work, we realized how fast the process could have been if there was a guide detailing the
 +
necessary resources and steps needed for a complete beginner in Rosetta. We figured that this might
 +
be one of the reasons why so few iGEM teams have used Rosetta in the past despite its vast capabilities.<br>
 +
<br>
 +
After getting great results from the software, we decided to share our experience and write this guide
 +
ourselves so that future iGEM teams can have a better starting point. We were delighted to find that
 +
we are not the only team using Rosetta this year and so we contacted iGEM Eindhoven and asked for
 +
their help with the guide. Their work on the guide – writing, sharing their protocols and experience
 +
was a valuable contribution that made the guide much more informative and comprehensive than we initially expected.
 +
<br>
 +
<br>
 +
<br>
 +
</p>
 +
</div>
 
</div>
 
</div>
 +
<div class="col-md-4 col-md-offset-4 col-sm-12 new-row">
 +
<a class="pop">
 +
<img src="https://static.igem.org/mediawiki/2016/c/ca/T--Technion_Israel--Rosetta_pic.png" class="img-responsive img-center" width="350" height="350" style="cursor: pointer;"><br>
 +
</a>
 +
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
−
</div><!-- END: #4 row -->
+
</div><!-- END: #1 row -->
 +
 
 +
 
 +
<br>
 
</div>
 
</div>
−
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<b>We processed potential variants of chemoreceptors for iGEM BGU using the Rosetta software.</b><br>
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We have used the Rosetta software in order to predict protein structure and to check for ligand-protein interactions.
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Running the software and processing both the ligand binding domain (LBD) of the Tar chemoreceptor from E. coli and
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the substances that interested iGEM BGU - Protocatechuic acid and Ethylene glycol, yielded a dozens of LBD variants.<br>
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After the filtering process with the desired filters a library of variants which should, theoretically, bind the
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ligands and induce an attraction chemotaxis reaction was obtained.<br>  
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1. CHONG, Shaorong; XU, Ming-Qun. Protein splicing of the Saccharomyces cerevisiae VMA intein without the endonuclease motifs. Journal of Biological Chemistry, 1997, 272.25: 15587-15590.‏ <br><br>
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2. ELLEUCHE, Skander; PÖGGELER, Stefanie. Inteins, valuable genetic elements in molecular biology and biotechnology. Applied Microbiology and Biotechnology, 2010, 87.2: 479-489.‏ <br><br>
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3. LIANG, Rubing; ZHOU, Jing; LIU, Jianhua. Construction of a bacterial assay for estrogen detection based on an estrogen-sensitive intein. Applied and environmental microbiology, 2011, 77.7: 2488-2495.‏ <br><br>
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4. Phyre2 modeling server <a href="http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index">online</a>. <br><br>
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5. SHIOMI, Daisuke, et al. Helical distribution of the bacterial chemoreceptor via colocalization with the Sec protein translocation machinery. Molecular microbiology, 2006, 60.4: 894-906.‏ <br><br>
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6. SKRETAS, Georgios; WOOD, David W. Regulation of protein activity with small‐molecule‐controlled inteins. Protein Science, 2005, 14.2: 523-532.‏ <br><br>
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7. TOPILINA, Natalya I.; MILLS, Kenneth V. Recent advances in in vivo applications of intein-mediated protein splicing. Mobile Dna, 2014, 5.1: 1.‏ <br><br>
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8. <a href="http://www.uniprot.org/uniprot/P03372">Uniprot‏</a><br>
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Revision as of 19:04, 12 October 2016

S.tar, by iGEM Technion 2016

S.tar, by iGEM Technion 2016


Peshawar – Tutoring the first Pakistani team

Introduction

We provided guidance to iGEM Peshawar, the first team to represent Pakistan. We assisted them with various aspects of Mathematical Modelling, Human Practice and cloning.

An opportunity for collaboration unveiled itself when we learned that the Pakistani team from Peshawar University is focusing on detection as a project. During the initial conversation we were amazed to learn that said group is the first Pakistani team to compete in iGEM, thus they were in need of guidance with most aspects of the competition. We decided to aid them and share all the knowledge and experience that was accumulated in our project and in past Technion iGEM teams. To achieve this, we used different platforms for communications mainly a Facebook group, where the Peshawar team could get an immediate answer to any question they had, and multiple Skype sessions each with a different focus such as mathematical modeling, Human practices, Cloning and more.


Mathematical modeling

We aided in the modeling process of Peshawar’s system, specifically using differential equations and logic gates. The aim of the model was to predict the expression of the reporter protein as a function of the inducers’ concentration.

View PDF


Cloning:

The Peshawar team had issues with the plasmid transformations into competent cells. After we got a full report on the exact steps they followed, we helped debugging their protocol.
We also shared our protocols of chemical transformation and competent cells preparation with them.
These instructions helped the team overcome their obstacles and proceed with the project.

Human practices:

We shared the layout of our educational program with Peshawar. Moreover, we advised the team regarding an appeal to the government with a request to promote the education in the field of Synthetic Biology.



Aachen- Prediction of mutated Subtilisin E protease

Introduction

We met with experts from the Technion to help us visualize the Subtilisin E and the photocaged serine in order to obtain insights regarding the structure of the mutated protein.

We contacted iGEM Aachen when we learned their project deals with protein inactivation, as we believed it might correspond with our Intein sub-project. After discussing the details we came to the conclusion that the Intein protein would not be applicable to Aachen's system. Nevertheless, we decided to collaborate on different aspects of our projects.

iGEM Aachen asked us to visualize and model the structure of the Subtilisin E protease, which is mutated with a photocaged serine as part of their project.
To do so we met with Prof. Meytal Landau (see: Attribution) from the faculty of Biology at the Technion who provided information regarding different visualization tools available to aid us in this.
As we tried to simulate the photocaged serine ourselves using the Chemdraw software we encountered a few obstacles. In order to overcome these obstacles, we contacted Einav Tayab-Fligelman, from Prof. Landau's lab, whom kindly provided us with a PDB file with a visualization of the Photocaged serine.
Furthermore, we contacted Dr. Fabian Glaser (see: Attribution) seeking assistance with the structure prediction. Dr. Glaser informed us that it would be impossible to receive a credible result within the short timeframe available. As an alternative he suggested writing a document describing the steps for an exhaustive computational work, including insights from the comparison of the structures of Subtilisin E and the photocaged serine.
Finally we created a 3D visualization of Subtilisin E adjacent to the photocaged serine along with the suggested document Dr. Glaser was kind enough to proofread and give his scientific remarks.

View PDF

The inevitable conclusion of the document was that the mutated Subtilisin E would not fold to the right tertiary structure. This information was highly valuable and helpful to Aachen’s project.


In return, we asked Aachen to build a biobrick consisting of the Tar chemoreceptor fused to a GFP marker. We provided them all the information necessary for this task.

View PDF

One of the greatest challenges in forming a fusion is finding the right linker which is meant to bridge between the proteins and assure that the proteins fold in a correct manner. Since we had some trouble finding the right linker, the Aachen team suggested to fuse the Tar to the GFP without a linker. The Tar (K777000) and the GFP (E0040) biobricks were obtained from the iGEM kit. The expression system constructed by the Aachen iGEM team, also consisted of an expression backbone- Promoter and RBS (J04500) and a terminator (B0015).


Fig. 1: The expression system which was constructed by the Aachen iGEM team, containing the Tar-GFP fusion. The current expression system does not include a linker.

After building the expression system with the Tar receptor fused to GFP, the plasmid was transformed to a BL21 E. coli strain. To validate the expression, the cloned bacteria were tested in a Tecan plate reader and under a fluorescent microscope.

GFP signal was detected on the polar part of the membrane only in a small fraction of cells. The majority of bacteria showed fluorescence in the entire volume of the cell, an indication of the accumulation of the Tar receptor inside the cell, probably due to an impaired structure of the protein (Fig. 2).



Fig. 2: Fluorescent microscope results.

The work made by Aachen has a major significance to our project and it gives decisive evidence to the importance of integrating a linker when fusing the proteins together. We would like to express our gratitude to the Aachen iGEM team for the valuable scientific work they have done for us.


TU Eindhoven - Writing a manual for the Rosetta software

Introduction

We have written a manual for the operation of the Rosetta software together with iGEM TU Eindhoven.

Our collaboration with iGEM Eindhoven was a result from the challenges we faced using the Rosetta software suite in our project. When we took our very first steps with protein modeling using Rosetta we quickly discovered numerous problems and difficulties that occupied us for weeks before we even started using the software.
During our work, we realized how fast the process could have been if there was a guide detailing the necessary resources and steps needed for a complete beginner in Rosetta. We figured that this might be one of the reasons why so few iGEM teams have used Rosetta in the past despite its vast capabilities.

After getting great results from the software, we decided to share our experience and write this guide ourselves so that future iGEM teams can have a better starting point. We were delighted to find that we are not the only team using Rosetta this year and so we contacted iGEM Eindhoven and asked for their help with the guide. Their work on the guide – writing, sharing their protocols and experience was a valuable contribution that made the guide much more informative and comprehensive than we initially expected.





BGU - Prediction of chemoreceptor-ligand interactions

Introduction

We processed potential variants of chemoreceptors for iGEM BGU using the Rosetta software.

We have used the Rosetta software in order to predict protein structure and to check for ligand-protein interactions. Running the software and processing both the ligand binding domain (LBD) of the Tar chemoreceptor from E. coli and the substances that interested iGEM BGU - Protocatechuic acid and Ethylene glycol, yielded a dozens of LBD variants.
After the filtering process with the desired filters a library of variants which should, theoretically, bind the ligands and induce an attraction chemotaxis reaction was obtained.



Referances

1. CHONG, Shaorong; XU, Ming-Qun. Protein splicing of the Saccharomyces cerevisiae VMA intein without the endonuclease motifs. Journal of Biological Chemistry, 1997, 272.25: 15587-15590.‏

2. ELLEUCHE, Skander; PÖGGELER, Stefanie. Inteins, valuable genetic elements in molecular biology and biotechnology. Applied Microbiology and Biotechnology, 2010, 87.2: 479-489.‏

3. LIANG, Rubing; ZHOU, Jing; LIU, Jianhua. Construction of a bacterial assay for estrogen detection based on an estrogen-sensitive intein. Applied and environmental microbiology, 2011, 77.7: 2488-2495.‏

4. Phyre2 modeling server online.

5. SHIOMI, Daisuke, et al. Helical distribution of the bacterial chemoreceptor via colocalization with the Sec protein translocation machinery. Molecular microbiology, 2006, 60.4: 894-906.‏

6. SKRETAS, Georgios; WOOD, David W. Regulation of protein activity with small‐molecule‐controlled inteins. Protein Science, 2005, 14.2: 523-532.‏

7. TOPILINA, Natalya I.; MILLS, Kenneth V. Recent advances in in vivo applications of intein-mediated protein splicing. Mobile Dna, 2014, 5.1: 1.‏

8. Uniprot‏



S.tar, by iGEM Technion 2016