Line 16: | Line 16: | ||
/* ==========Background and effects ========== */ | /* ==========Background and effects ========== */ | ||
/*Make sure the div is unuiqe to each page*/ | /*Make sure the div is unuiqe to each page*/ | ||
− | . | + | .intein_wrapper { |
position: relative; | position: relative; | ||
background: white; /* For browsers that do not support gradients */ | background: white; /* For browsers that do not support gradients */ | ||
− | background: -webkit-linear-gradient(white, # | + | background: -webkit-linear-gradient(white, #ecf7fb, white); /* For Safari 5.1 to 6.0 */ |
− | background: -o-linear-gradient(white, # | + | background: -o-linear-gradient(white, #ecf7fb, white); /* For Opera 11.1 to 12.0 */ |
− | background: -moz-linear-gradient(white, # | + | background: -moz-linear-gradient(white, #ecf7fb, white); /* For Firefox 3.6 to 15 */ |
− | background: linear-gradient(white, # | + | background: linear-gradient(white, #ecf7fb, white); /* Standard syntax */ |
/*background-image:url('addres'); | /*background-image:url('addres'); | ||
background-size:cover; | background-size:cover; | ||
Line 29: | Line 29: | ||
} | } | ||
/* ========== END: and effects ========== */ | /* ========== END: and effects ========== */ | ||
+ | |||
+ | .referances { | ||
+ | font-size: 16px; | ||
+ | outline: 1px solid black; | ||
+ | padding: 20px; | ||
+ | } | ||
Line 50: | Line 56: | ||
*/ | */ | ||
+ | /*canceling wiki bug (inlarge imgs stuck the page)*/ | ||
+ | .modal-backdrop { | ||
+ | position: relative; | ||
+ | } | ||
</style> | </style> | ||
Line 77: | Line 87: | ||
}); | }); | ||
+ | //////////////////////// | ||
+ | //Code: inlarge img on click: | ||
+ | $(function() { | ||
+ | $('.pop').on('click', function() { | ||
+ | $('.imagepreview').attr('src', $(this).find('img').attr('src')); | ||
+ | $('#imagemodal').modal('show'); | ||
+ | }); | ||
+ | }); | ||
+ | ////////////////////// | ||
+ | ///pop-ups: | ||
+ | $(document).ready(function(){ | ||
+ | $('[data-toggle="popover"]').popover(); | ||
+ | }); | ||
+ | |||
+ | $(function () { | ||
+ | $('button').popover(); | ||
+ | }); | ||
+ | |||
+ | /////////////////////// | ||
+ | |||
</script> | </script> | ||
Line 89: | Line 119: | ||
<!-- ===== One overall container (wrapper) ===== --> | <!-- ===== One overall container (wrapper) ===== --> | ||
− | <div class=" | + | <div class="intein_wrapper"> |
Line 116: | Line 146: | ||
<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> | + | <br><h4 class="text-center"><b>Peshawar</b></h4> |
</a> | </a> | ||
</li> | </li> | ||
Line 123: | Line 153: | ||
<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> | + | <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="# | + | <a href="#Results" aria-controls="Results" role="tab" data-toggle="tab"> |
− | <img src="https://static.igem.org/mediawiki/2016/ | + | <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> | + | <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="# | + | <a href="#outlook" aria-controls="outlook" role="tab" data-toggle="tab"> |
− | <img src="https://static.igem.org/mediawiki/2016/ | + | <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> | + | <br><h4 class="text-center"><b>BGU</b></h4> |
</a> | </a> | ||
</li> | </li> | ||
Line 145: | Line 175: | ||
</div> | </div> | ||
<!-- ========== End: Tabs panel ========== --> | <!-- ========== End: Tabs panel ========== --> | ||
+ | |||
+ | |||
+ | |||
+ | |||
Line 150: | Line 184: | ||
<div class="tab-content"> | <div class="tab-content"> | ||
− | <!-- =========== Peshawar =========== --> | + | |
− | <div role="tabpanel" class="tab-pane fade | + | |
+ | |||
+ | <!-- =========== 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> | + | <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 --> | ||
Line 168: | Line 202: | ||
<div class="cont_box"> | <div class="cont_box"> | ||
<div class="row"> | <div class="row"> | ||
− | |||
<div class="col-sm-12"> | <div class="col-sm-12"> | ||
− | <div class="col-md- | + | <div class="col-md-12 col-sm-12"> |
− | <h2 class=""> | + | <h2 class="">Introduction</h2> |
<p class="text-justify"> | <p class="text-justify"> | ||
− | + | <b>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.</b><br> | |
− | them the knowledge that was accumulated | + | <br> |
− | group, | + | 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. | ||
</p> | </p> | ||
</div> | </div> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
</div> | </div> | ||
</div> | </div> | ||
Line 198: | Line 232: | ||
<div class="cont_box"> | <div class="cont_box"> | ||
<div class="row"> | <div class="row"> | ||
− | |||
<div class="col-sm-12"> | <div class="col-sm-12"> | ||
− | <div class="col-md | + | <div class="col-md-12 col-sm-12"> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
<h2 class="">Mathematical modeling</h2> | <h2 class="">Mathematical modeling</h2> | ||
<p class="text-justify"> | <p class="text-justify"> | ||
− | + | 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.<br> | |
− | < | + | </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> | ||
Line 221: | Line 252: | ||
<br> | <br> | ||
+ | |||
<div class="row"><!-- #3 row --> | <div class="row"><!-- #3 row --> | ||
Line 226: | Line 258: | ||
<div class="cont_box"> | <div class="cont_box"> | ||
<div class="row"> | <div class="row"> | ||
− | |||
<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=""> | + | <h2 class="">Cloning:</h2> |
<p class="text-justify"> | <p class="text-justify"> | ||
− | + | 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.<br> | |
− | + | 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 | + | |
− | < | + | <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> | ||
Line 249: | Line 286: | ||
<br> | <br> | ||
+ | </div> | ||
+ | <!-- =========== END: Intro =========== --> | ||
− | <div class="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"> | ||
− | + | <div class="col-md-12 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- | + | <p class="text-center"> |
− | <img src=" | + | <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- | + | <div class="col-md-12 col-sm-12 new-row no-title-col"> |
− | + | ||
<p class="text-justify"> | <p class="text-justify"> | ||
− | + | 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.<br> | |
− | + | <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: # | + | </div><!-- END: #1 row --> |
+ | |||
+ | |||
+ | <br> | ||
</div> | </div> | ||
− | <!-- =========== END: | + | <!-- ====================END: 3 TU Eindhoven ==================== --> |
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | <div role="tabpanel" class="tab-pane fade" id=" | + | |
− | + | <!-- ==================== 4: BGU ==================== --> | |
− | < | + | <div role="tabpanel" class="tab-pane fade" id="outlook"> |
− | + | <div class="row"> <!--Headline--> | |
− | < | + | <div class="col-sm-12"> |
− | + | <br> | |
− | + | <h1 class="text-center"><u>BGU - Prediction of chemoreceptor-ligand interactions</u></h1> | |
− | 1 | + | </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 processed potential variants of chemoreceptors for iGEM BGU using the Rosetta software.</b><br> | ||
+ | <br> | ||
+ | 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.<br> | ||
+ | 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.<br> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div><!-- END: #1 row --> | ||
</div> | </div> | ||
+ | <!-- ==================== END: 4 BGU ==================== --> | ||
</div><!-- ============ END: Tabs ============ --> | </div><!-- ============ END: Tabs ============ --> | ||
+ | |||
+ | |||
+ | <!--Code: Click on img to enlarge it--> | ||
+ | <div class="modal fade" id="imagemodal" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true"> | ||
+ | <div class="modal-dialog"> | ||
+ | <div class="modal-content"> | ||
+ | <div class="modal-body"> | ||
+ | <button type="button" class="close" data-dismiss="modal"><span aria-hidden="true">×</span><span class="sr-only">Close</span></button> | ||
+ | <img src="" class="imagepreview" style="width: 100%;" > | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
</div> | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <br> | ||
+ | <br> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="row"> | ||
+ | <div class="col-sm-10 col-sm-offset-1"> | ||
+ | <a href="#intein_referances" data-toggle="collapse">Referances</a> | ||
+ | <div id="intein_referances" class="collapse"> | ||
+ | |||
+ | <p class="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. <br><br> | ||
+ | 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> | ||
+ | 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> | ||
+ | 4. Phyre2 modeling server <a href="http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index">online</a>. <br><br> | ||
+ | 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> | ||
+ | 6. SKRETAS, Georgios; WOOD, David W. Regulation of protein activity with small‐molecule‐controlled inteins. Protein Science, 2005, 14.2: 523-532. <br><br> | ||
+ | 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> | ||
+ | 8. <a href="http://www.uniprot.org/uniprot/P03372">Uniprot</a><br> | ||
+ | </p> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <br> | ||
+ | <br> | ||
+ | |||
<a id="back-to-top" href="#" class="btn btn-lg back-to-top" role="button" title="Up" data-toggle="tooltip" data-placement="left"><img src="https://static.igem.org/mediawiki/2016/5/5a/T--Technion_Israel--up_arrow.png" alt=""></a> | <a id="back-to-top" href="#" class="btn btn-lg back-to-top" role="button" title="Up" data-toggle="tooltip" data-placement="left"><img src="https://static.igem.org/mediawiki/2016/5/5a/T--Technion_Israel--up_arrow.png" alt=""></a> |
Revision as of 19:04, 12 October 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.
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.
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.
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.
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