|
|
| Line 362: |
Line 362: |
| | | | |
| | <br> | | <br> |
| − | <br>
| |
| − | <b class="bg-danger">====================** Video #1 **====================</b>
| |
| | </p> | | </p> |
| | <!-- 12 img div --> | | <!-- 12 img div --> |
| Line 386: |
Line 384: |
| | </div> | | </div> |
| | </div> | | </div> |
| − | <br> | + | |
| − | <br>
| + | |
| − | | + | |
| | <p class="text-justify"> | | <p class="text-justify"> |
| | In attempts to understand and repeat this phenomenon, a range of estradiol concentrations was tested but none of them succeeded.</p> | | In attempts to understand and repeat this phenomenon, a range of estradiol concentrations was tested but none of them succeeded.</p> |
| Line 398: |
Line 394: |
| | <b class="bg-danger">====================** Video #2 **====================</b> | | <b class="bg-danger">====================** Video #2 **====================</b> |
| | </p> | | </p> |
| − | | + | <div class="row"> |
| − | | + | <div class="col-md-4 col-sm-12"> |
| | + | <p class="text-justify">a.</p> |
| | + | <video autoplay loop class="embed-responsive-item"> |
| | + | <source src="https://static.igem.org/mediawiki/2016/6/61/T--Technion_Israel--est_video_2a.mp4" type="video/mp4"> |
| | + | </video> |
| | + | </div> |
| | + | </div> |
| | + | <div class="row"> |
| | + | <div class="row"> |
| | + | <div class="col-md-4 col-sm-12"> |
| | + | <p class="text-justify">b.</p> |
| | + | <video autoplay loop class="embed-responsive-item"> |
| | + | <source src="https://static.igem.org/mediawiki/2016/9/97/T--Technion_Israel--est_video_2b.mp4" type="video/mp4"> |
| | + | </video> |
| | + | </div> |
| | + | </div> |
| | + | <div class="row"> |
| | + | <div class="col-md-12 col-sm-12"> |
| | + | |
| | <b>Video 2:</b> <a href="https://2016.igem.org/Team:Technion_Israel/Experiments" ><b>microscope assay</b></a> | | <b>Video 2:</b> <a href="https://2016.igem.org/Team:Technion_Israel/Experiments" ><b>microscope assay</b></a> |
| | <b>a.</b> Adding 10<sup>-1</sup>[mg/ml] 17-β-estradiol in DMSO to | | <b>a.</b> Adding 10<sup>-1</sup>[mg/ml] 17-β-estradiol in DMSO to |
| Line 432: |
Line 446: |
| | UERT<i class="entypo-check"></i></button></a>. | | UERT<i class="entypo-check"></i></button></a>. |
| | </p> | | </p> |
| − | | + | </div> |
| − |
| + | |
| − | | + | |
| − | | + | |
| − | </div>
| + | |
| − | | + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div><!-- END: #1 row -->
| + | |
| − | </div>
| + | |
| − | <!-- ====================END: 3 Resuls ==================== -->
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <!-- ==================== 4: Outlook ==================== -->
| + | |
| − | <div role="tabpanel" class="tab-pane fade" id="outlook">
| + | |
| − | <div class="row"> <!--Headline-->
| + | |
| − | <div class="col-sm-12">
| + | |
| − | <br>
| + | |
| − | <h1 class="text-center"><u>Outlook</u></h1>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <!-- ========== Content ========== -->
| + | |
| − | | + | |
| − | | + | |
| − | <div class="row"><!-- #1 row -->
| + | |
| − | <div class="col-sm-8 col-sm-offset-2"><!-- 8/12 -->
| + | |
| − | | + | |
| − | <div class="cont_box">
| + | |
| − | <div class="row">
| + | |
| − | <div class="col-sm-12">
| + | |
| − | | + | |
| − | <p class="text-justify">
| + | |
| − | Although the concept of this idea seemed promising, but the PctA-Tar and NarX chimeras had more potential to succeed, due to their structure similarity to the native Tar chemoreceptor (they all contain
| + | |
| − | <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true" data-content="an approximately 50 amino acid region that connect extracellular sensory with intracellular signaling domains in over 7500 proteins, including histidine kinases, adenylyl cyclases, chemotaxis receptors, and phosphatases).">
| + | |
| − | HAMP<i class="entypo-check"></i></button></a>
| + | |
| − | domain, which is located invariably at the C-terminal end of the last transmembrane segment). Both mentioned chimeras were built using their own HAMP, rather than Tars’ native HAMP. In contrast, the hERɑ does not contain HAMP region, thus its chimera was built using the Tar’s native HAMP. We believe that this is the main reason of the chimera failure, and results of all tests support this assumption. For an example, in the modeled 3D structure, it is clear that HAMP region is the problematic, as there it is clear the HAMP is out of place.
| + | |
| − | Furthermore, the results of the GFP also fit this assumption, as the chimera was unsuccessful at crossing the membrane, probable due to the HAMP.
| + | |
| − | Lastly, most of the reported chemotaxis receptors naturally contain a HAMP domain, and seems to important in regulating the coiled-coil interactions that seem to be involved in signal propagation, which further supports our assumption <b>(2,5)</b>.<br><br><br>
| + | |
| − |
| + | |
| − | To conclude, further research is needed in order to fully understand the cause of failure and overcome the obstacles faced in this attempt of generating a new chemoreceptor which reacts to estrogen derivatives.
| + | |
| − | The main problems and a way to overcome them are as follows:<br>
| + | |
| − | * The estrogen derivatives used were solubile only in hydrophics solvents, which are lethal to the bacteria. The use of estrogen soluble in water or other non- lethal solvents should solve this problem.<br>
| + | |
| − | * The 3D model did indicated incorrect folding of the newly designed receptor. A new design focused more on the Tar might aid in solving this problem.<br>
| + | |
| − | * The hERα does not naturally contain a HAMP region. Using a chemoreceptor that does have a HAMP domain might help overcoming this obstacle.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div><!-- END: #1 row -->
| + | |
| − | </div>
| + | |
| − | <!-- ==================== END: 4 Outlook ==================== -->
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | </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-8 col-sm-offset-2"><!-- 8/12 -->
| |
| − |
| |
| − |
| |
| − |
| |
| − | <p class="references">
| |
| − | References:<br>
| |
| − | 1. CHEN, Dongsheng, et al. Phosphorylation of human estrogen receptor α by protein kinase A regulates dimerization. Molecular and cellular biology, 1999, 19.2: 1002-1015.<br><br>
| |
| − | 2. HULKO, Michael, et al. The HAMP domain structure implies helix rotation in transmembrane signaling. Cell, 2006, 126.5: 929-940. <br><br>
| |
| − | 3. <a href="http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index">Phyre2 modeling server</a>.<br><br>
| |
| − | 4. 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>
| |
| − | 5. WADHAMS, George H.; ARMITAGE, Judith P. Making sense of it all: bacterial chemotaxis. Nature Reviews Molecular Cell Biology, 2004, 5.12: 1024-1037.<br><br>
| |
| − |
| |
| − | </p>
| |
| − |
| |
| − |
| |
| − | </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>
| |
| − |
| |
| − |
| |
| − | </body>
| |
| − | </html>
| |
| − | {{:Team:Technion_Israel/supporters}}
| |
| − | {{Team:Technion_Israel/navbar}}
| |
| − | <html lang="en">
| |
| − |
| |
| − | <head>
| |
| − | <!-- Title for the page & metadata -->
| |
| − | <title>S.tar, by iGEM Technion 2016</title>
| |
| − | <meta charset="utf-8">
| |
| − | <meta name="navbar" content="width=device-width, initial-scale=1">
| |
| − |
| |
| − |
| |
| − | <style>/* inline page CSS */
| |
| − |
| |
| − | body {
| |
| − | }
| |
| − |
| |
| − | /* ==========Background and effects ========== */
| |
| − | /*Make sure the div is unuiqe to each page*/
| |
| − | .intein_wrapper {
| |
| − | position: relative;
| |
| − |
| |
| − | background: white; /* For browsers that do not support gradients */
| |
| − | background: -webkit-linear-gradient(white, #f8eee2, white); /* For Safari 5.1 to 6.0 */
| |
| − | background: -o-linear-gradient(white, #f8eee2, white); /* For Opera 11.1 to 12.0 */
| |
| − | background: -moz-linear-gradient(white, #f8eee2, white); /* For Firefox 3.6 to 15 */
| |
| − | background: linear-gradient(white, #f8eee2, white); /* Standard syntax */
| |
| − | /*background-image:url('addres');
| |
| − | background-size:cover;
| |
| − | filter: blur(5px) grayscale(80%) opacity(10%);*/
| |
| − | }
| |
| − | /* ========== END: and effects ========== */
| |
| − |
| |
| − | .references {
| |
| − | font-size: 16px;
| |
| − | outline: 1px solid black;
| |
| − | padding: 20px;
| |
| − | }
| |
| − |
| |
| − |
| |
| − | /*
| |
| − | Object in external CSS sheet:
| |
| − |
| |
| − | nav-tabs, cont_tabs:
| |
| − | Open diffrent tabs, we uses imgs.
| |
| − |
| |
| − | back-to-top:
| |
| − | go back to top. Apears only when going down the page.
| |
| − |
| |
| − | cont_box:
| |
| − | The containers (box) which hold the texts and imgs in the page.
| |
| − |
| |
| − | img_cont:
| |
| − | Every in-content-page img needs to have this class of img.
| |
| − |
| |
| − | no-title-col:
| |
| − | Every in-content-page img needs to have this class of col.
| |
| − | */
| |
| − |
| |
| − | /*canceling wiki bug (inlarge imgs stuck the page)*/
| |
| − | .modal-backdrop {
| |
| − | position: relative;
| |
| − | }
| |
| − |
| |
| − | </style>
| |
| − |
| |
| − |
| |
| − | <script>
| |
| − |
| |
| − | //up arrow:
| |
| − | $(document).ready(function(){
| |
| − | $(window).scroll(function () {
| |
| − | if ($(this).scrollTop() > 350) {
| |
| − | $('#back-to-top').fadeIn();
| |
| − | } else {
| |
| − | $('#back-to-top').fadeOut();
| |
| − | }
| |
| − | });
| |
| − | // scroll body to 0px on click
| |
| − | $('#back-to-top').click(function () {
| |
| − | $('#back-to-top').tooltip('hide');
| |
| − | $('body,html').animate({
| |
| − | scrollTop: 0
| |
| − | }, 800);
| |
| − | return false;
| |
| − | });
| |
| − |
| |
| − | $('#back-to-top').tooltip('show');
| |
| − |
| |
| − | });
| |
| − | ////////////////////////
| |
| − | //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>
| |
| − |
| |
| − | </head>
| |
| − |
| |
| − |
| |
| − | <body>
| |
| − |
| |
| − | <header>
| |
| − | </header>
| |
| − |
| |
| − | <!-- ===== One overall container (wrapper) ===== -->
| |
| − | <div class="intein_wrapper">
| |
| − |
| |
| − |
| |
| − |
| |
| − | <!-- ======== Cover photo: ======== -->
| |
| − | <div class="row">
| |
| − | <div class="col-xs-12">
| |
| − | <img src="https://static.igem.org/mediawiki/2016/9/93/T--Technion_Israel--hERacov.jpg" class="img-responsive img-center cont_cover" width="100%">
| |
| − | </div>
| |
| − | </div>
| |
| − | <!-- ===== END: Cover photo: ===== -->
| |
| − |
| |
| − |
| |
| − | <br>
| |
| − |
| |
| − |
| |
| − | <!-- ============ Tabs: ============ -->
| |
| − |
| |
| − | <!-- =============== Tabs names: =============== -->
| |
| − | <div class="row">
| |
| − | <div class="col-sm-8 col-sm-offset-2"><!-- 8/12 -->
| |
| − |
| |
| − | <ul class="nav nav-tabs" role="tablist">
| |
| − |
| |
| − | <li role="presentation" class="col-sm-3 col-xs-6">
| |
| − | <a href="#Peshawar" aria-controls="Peshawar" role="tab" data-toggle="tab">
| |
| − | <img src="https://static.igem.org/mediawiki/2016/d/db/T--Technion_Israel--icon_intro.png" class="img-responsive img-center cont_tabs" width="75" height="75">
| |
| − | <br><h4 class="text-center"><b>Introduction</b></h4>
| |
| − | </a>
| |
| − | </li>
| |
| − |
| |
| − | <li role="presentation" class="col-sm-3 col-xs-6">
| |
| − | <a href="#Lab" aria-controls="Lab" role="tab" data-toggle="tab">
| |
| − | <img src="https://static.igem.org/mediawiki/2016/4/49/T--Technion_Israel--icon_lab.png" class="img-responsive img-center cont_tabs" width="75" height="75">
| |
| − | <br><h4 class="text-center"><b>Design and Implementation</b></h4>
| |
| − | </a>
| |
| − | </li>
| |
| − |
| |
| − | <li role="presentation" class="col-sm-3 col-xs-6">
| |
| − | <a href="#Results" aria-controls="Results" role="tab" data-toggle="tab">
| |
| − | <img src="https://static.igem.org/mediawiki/2016/4/45/T--Technion_Israel--icon_results.png" class="img-responsive img-center cont_tabs" width="75" height="75">
| |
| − | <br><h4 class="text-center"><b>Results</b></h4>
| |
| − | </a>
| |
| − | </li>
| |
| − |
| |
| − | <li role="presentation" class="col-sm-3 col-xs-6">
| |
| − | <a href="#outlook" aria-controls="outlook" role="tab" data-toggle="tab">
| |
| − | <img src="https://static.igem.org/mediawiki/2016/4/47/T--Technion_Israel--icon_outlook.png" class="img-responsive img-center cont_tabs" width="75" height="75">
| |
| − | <br><h4 class="text-center"><b>Outlook</b></h4>
| |
| − | </a>
| |
| − | </li>
| |
| − |
| |
| − | </ul>
| |
| − | </div>
| |
| − | </div>
| |
| − | <!-- ========== End: Tabs panel ========== -->
| |
| − |
| |
| − |
| |
| − | <!-- =========== Tabs' content =========== -->
| |
| − | <div class="tab-content">
| |
| − |
| |
| − | <!-- =========== 1. Intro =========== -->
| |
| − | <div role="tabpanel" class="tab-pane fade in active" id="Peshawar">
| |
| − | <div class="row"> <!--Headline-->
| |
| − | <div class="col-sm-12">
| |
| − | <br>
| |
| − | <h1 class="text-center"><u>Introduction</u></h1>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <!-- ========== Content ========== -->
| |
| − |
| |
| − |
| |
| − | <div class="row"><!-- #1 row -->
| |
| − | <div class="col-sm-8 col-sm-offset-2"><!-- 8/12 -->
| |
| − |
| |
| − | <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">
| |
| − | Our attempts to fuse two segments originating from different organisms to design a new receptor was met with great challenges. These specific segments were the LBD of the Human Estrogen Receptor α (hERα) and the cytoplasmic domain of Tar.<br><br>
| |
| − |
| |
| − |
| |
| − | <b>hERɑ</b> is a human nuclear receptor that induces signal transduction in response to estrogenic compounds. Despite the fact that bacterial chemoreceptors are comprised of a two component system and the hERα is not, we assumed that hERa will trigger the phosphorylation cascade of the chemotaxis system, due to the conformational changes caused by the estrogen binding to its domain. This led us to design and construct the new hybrid: hERα-Tar <b> (1)</b>. <b> (Add an image T--Technion_Israel--hERa_receptor_Fig1)<b>
| |
| − |
| |
| − |
| |
| − |
| |
| − | </p>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div><!-- END: #1 row -->
| |
| − | </div>
| |
| − | <!-- =========== END: Intro =========== -->
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − | <!-- =========== 2: conection to the project =========== -->
| |
| − | <div role="tabpanel" class="tab-pane fade" id="Lab">
| |
| − | <div class="row"> <!--Headline-->
| |
| − | <div class="col-sm-12">
| |
| − | <br>
| |
| − | <h1 class="text-center"><u>Procedure:</u></h1>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <!-- ========== Content ========== -->
| |
| − |
| |
| − |
| |
| − | <div class="row"><!-- #1 row -->
| |
| − | <div class="col-sm-8 col-sm-offset-2"><!-- 8/12 -->
| |
| − |
| |
| − | <div class="cont_box">
| |
| − | <div class="row">
| |
| − | <div class="col-sm-12">
| |
| − |
| |
| − | <p class="text-justify">
| |
| − | The <a href="https://2016.igem.org/Team:Technion_Israel/Modifications/Intein" >intein-gBlock</a> was designed with the estrogen
| |
| − | <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
| |
| − | data-content="Ligand Binding Domain">
| |
| − | LBD<i class="entypo-check"></i></button></a>
| |
| − | site as the splicing inducer. The cDNA sequence was the source of said <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
| |
| − | data-content="Ligand Binding Domain">
| |
| − | LBD<i class="entypo-check"></i></button></a> in the intein gBlock.
| |
| − | This design provided the team an opportunity to easily extract the <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
| |
| − | data-content="Ligand Binding Domain">
| |
| − | LBD<i class="entypo-check"></i></button></a> and fuse it to
| |
| − | <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true" data-content="An approximately 50 amino acid region that connect extracellular sensory with intracellular signaling domains in over 7500 proteins, including histidine kinases, adenylyl cyclases, chemotaxis receptors, and phosphatases.">
| |
| − | HAMP<i class="entypo-check"></i></button></a>
| |
| − | domain, which is located at the <br>C-terminal end of the last transmembrane segment of Tar, to get a final hybrid product hERα-Tar.
| |
| − | The new chimera was cloned to <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true" data-content="An <i>E.coli</i> derivative, which lacks chemoreceptors genes, means this strain does not obtain chemotaxis ability."> UU1250<i class="entypo-check"></i></button></a> to generate the new strain: <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true" data-content="UU1250 cloned with hERɑ-Tar chimera">UERT<i class="entypo-check"></i></button></a>. To the best of our knowledge, this design and cloning has never been reported before.<br>
| |
| − |
| |
| − | <b> Add image: T--Technion Israel--estrogencircute..
| |
| − | Fig. 2: The hERɑ-Tar chimera circuit. </b>
| |
| − |
| |
| − | <br>
| |
| − | In order to predict the feasibility of this new hybrid, a 3D model was made using the Phyre2 Fold Recognition server <b>(3)</b>.
| |
| − | Later, in order to confirm the correct localization at both poles of the bacterial membrane <b>(4)</b>, a GFP reporter protein was fused to the hERα-Tar chimera and tested with fluorescence microscopy.<br>
| |
| − |
| |
| − | <br>
| |
| − | Finally, a “Chip Microscope assay” was conducted to study the effects of 17- β-estradiol on the chemotaxis system of the <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true" data-content="UU1250 cloned with hERɑ-Tar chimera">
| |
| − | UERT<i class="entypo-check"></i></button></a> strain. In short, a suspension of the <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true" data-content="UU1250 cloned with hERɑ-Tar chimera">
| |
| − | UERT<i class="entypo-check"></i></button></a> strain was added to an ibidi microchannel chip, and the bacterial concentration was monitored in a fixed point for the whole experiment, as the estradiol was added to the channel.
| |
| − | </p>
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div><!-- END: #1 row -->
| |
| − | </div>
| |
| − | <!-- =========== END: 2 - conection to the project =========== -->
| |
| − |
| |
| − |
| |
| − |
| |
| − | <!-- ==================== 3: Resuls ==================== -->
| |
| − | <div role="tabpanel" class="tab-pane fade" id="Results">
| |
| − |
| |
| − | <div class="row"> <!--Headline-->
| |
| − | <div class="col-sm-12">
| |
| − | <br>
| |
| − | <h1 class="text-center"><u>Results</u></h1>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <!-- ========== Content ========== -->
| |
| − |
| |
| − |
| |
| − | <div class="row"><!-- #1 row -->
| |
| − | <div class="col-sm-8 col-sm-offset-2"><!-- 8/12 -->
| |
| − |
| |
| − | <div class="cont_box">
| |
| − | <div class="row">
| |
| − | <div class="col-sm-12">
| |
| − |
| |
| − | <p class="text-justify">
| |
| − | The 3D structure of the hERα-Tar, as can be seen in figure 3, clearly indicates an incorrect folding of the
| |
| − | <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true" data-content="an approximately 50 amino acid region that connect extracellular sensory with intracellular signaling domains in over 7500 proteins, including histidine kinases, adenylyl cyclases, chemotaxis receptors, and phosphatases.">
| |
| − | HAMP<i class="entypo-check"></i></button></a>
| |
| − | region, and thus an overall incorrect structure. Nevertheless, the rest of the tests were conducted in hope for successful results.
| |
| − | </p><br>
| |
| − | <div class="col-md-6 col-sm-12">
| |
| − | <p class="text-justify">a.</p>
| |
| − | <a class="pop">
| |
| − | <img src="https://static.igem.org/mediawiki/2016/thumb/0/06/T--Technion_Israel--ESTR_tar_fig_1a.PNG/800px-T--Technion_Israel--ESTR_tar_fig_1a.PNG" class="img-responsive img-center img-cont" width="400" style="cursor: pointer;"><br>
| |
| − | </a>
| |
| − | </div>
| |
| − | <div class="col-md-6 col-sm-12">
| |
| − | <p class="text-justify">b.</p>
| |
| − | <a class="pop">
| |
| − | <img src="https://static.igem.org/mediawiki/2016/thumb/f/f2/T--Technion_Israel--figure6c.png/799px-T--Technion_Israel--figure6c.png" class="img-responsive img-center img-cont" width="400" style="cursor: pointer;"><br>
| |
| − | </a>
| |
| − | </div>
| |
| − | <b>Fig.3 :</b> <b>a.</b> a model demonstration of a predict structure of hERɑ-Tar chimera. <b>b.</b> a model demonstration of a predict structure of Tar chemoreceptor. </b>
| |
| − | <p class="text-justify">
| |
| − | The results of the fluorescence microscopy were not promising neither. That is due to the fact that although the GFP was expressed, the signal indicated that the chimera failed to localize at the poles and stayed in the cytoplasm, figure 4. In other words, in case that the chimera is expressed it will not be able to cross the membrane, and thus the chemotaxis system will not function correctly.
| |
| | | | |
| − | </p><br>
| |
| − |
| |
| − |
| |
| − | <div class="col-md-6 col-sm-12">
| |
| − | <p class="text-justify">a.</p>
| |
| − | <a class="pop">
| |
| − | <img src="https://static.igem.org/mediawiki/2016/5/5d/T--Technion_Israel--estrotar_figure2a.jpg" class="img-responsive img-center img-cont" width="400" style="cursor: pointer;"><br>
| |
| − | </a>
| |
| − | </div>
| |
| − |
| |
| − | <div class="col-md-6 col-sm-12">
| |
| − | <p class="text-justify">b.</p>
| |
| − | <a class="pop">
| |
| − | <img src="https://static.igem.org/mediawiki/2016/9/9c/T--Technion_Israel--estrotar_figure2b.jpg" class="img-responsive img-center img-cont" width="400" style="cursor: pointer;"><br>
| |
| − | </a>
| |
| − | </div>
| |
| − |
| |
| − | <div class="col-md-12 col-sm-12">
| |
| − | <p class="text-justify">
| |
| − | <b>Fig. 4:</b>
| |
| − | <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
| |
| − | data-content="UU1250 cloned with hERɑ-Tar chimera fused with GFP">
| |
| − | UERTG<i class="entypo-check"></i></button></a>
| |
| − | under a fluorescence microscope. <b>a.</b> Under fluorescence light at 490nm excitation. <b>b.</b> Under white light.
| |
| − | </p>
| |
| − |
| |
| − | </div>
| |
| − | <div class="col-md-12 col-sm-12">
| |
| − | <p class="text-justify">
| |
| − | <br>
| |
| − | Despite the discouraging results from the GFP assay we attempted the “Chip microscope assay” to test for any real time response. Our first tests ended abruptly as we discovered that the estrogenic solution kills the bacteria almost immediately. <br>Later, we concluded that the fault was in the solvent that was used to dissolve the compound, 17-β-estradiol. Since this is a hydrophobic substance, it is only possible to dissolve it in hydrophobic solvents, such as Ethanol and DMSO, which are lethal for bacteria. When the stock solution of 17-β-estradiol in DMSO, was diluted to a concentration that was not lethal for the bacteria - 0.1% DMSO content, no signs of response were visible.<br><br>
| |
| − | A noteworthy phenomenon that occurred during one of the microscope tests, was when a solution of 17-β-estradiol dissolved in DMSO (concentration 10<sup>-5</sup> mg/ml) was added to the <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
| |
| − | data-content="UU1250 cloned with hERɑ-Tar chimera">
| |
| − | UERT<i class="entypo-check"></i></button></a> strain suspension, and completely halted the bacterial movement, but several minutes later the bacteria regained viability. This did not occur when tried on the control strain.
| |
| − |
| |
| − |
| |
| − | <br>
| |
| − | <br>
| |
| − | <b class="bg-danger">====================** Video #1 **====================</b>
| |
| − | </p>
| |
| − | <!-- 12 img div -->
| |
| − | <div class="row">
| |
| − | <div class="col-md-12 col-sm-12">
| |
| − | <video autoplay loop class="embed-responsive-item">
| |
| − | <source src="https://static.igem.org/mediawiki/2016/8/8e/T--Technion_Israel--est_video_1a.mp4 " type="video/mp4">
| |
| − | </video>
| |
| − |
| |
| − | <p class="text-center">
| |
| − | <b>Video 1:</b>
| |
| − | <a href="https://2016.igem.org/Team:Technion_Israel/Experiments" ><b>microscope assay</b></a>.
| |
| − | <b>a.</b> Adding 10<sup>-5</sup>[mg/ml] 17-β-estradiol in DMSO to
| |
| − | <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
| |
| − | data-content="UU1250 cloned with hERɑ-Tar chimera">
| |
| − | UERT<i class="entypo-check"></i></button></a>.
| |
| − | <b>b.</b> Control - Adding DMSO to
| |
| − | <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
| |
| − | data-content="UU1250 cloned with hERɑ-Tar chimera">
| |
| − | UERT<i class="entypo-check"></i></button></a>.
| |
| − | </p>
| |
| − | </div>
| |
| − | </div>
| |
| − | <br>
| |
| − | <br>
| |
| − |
| |
| − | <p class="text-justify">
| |
| − | In attempts to understand and repeat this phenomenon, a range of estradiol concentrations was tested but none of them succeeded.</p>
| |
| − |
| |
| − | <br>
| |
| − | <br>
| |
| − |
| |
| − | <p class="text-justify">
| |
| − | <b class="bg-danger">====================** Video #2 **====================</b>
| |
| − | </p>
| |
| − |
| |
| − | <p class="text-justify">
| |
| − | <b>Video 2:</b> <a href="https://2016.igem.org/Team:Technion_Israel/Experiments" ><b>microscope assay</b></a>
| |
| − | <b>a.</b> Adding 10<sup>-1</sup>[mg/ml] 17-β-estradiol in DMSO to
| |
| − | <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
| |
| − | data-content="UU1250 cloned with hERɑ-Tar chimera">
| |
| − | UERT<i class="entypo-check"></i></button></a>.
| |
| − |
| |
| − | <b>b.</b> Adding 10<sup>-2</sup>[mg/ml] 17-β-estradiol in DMSO to
| |
| − | <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
| |
| − | data-content="UU1250 cloned with hERɑ-Tar chimera">
| |
| − | UERT<i class="entypo-check"></i></button></a>.
| |
| − |
| |
| − | <br><b>c.</b> Adding 10<sup>-3</sup>[mg/ml] 17-β-estradiol in DMSO to
| |
| − | <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
| |
| − | data-content="UU1250 cloned with hERɑ-Tar chimera">
| |
| − | UERT<i class="entypo-check"></i></button></a>.
| |
| − |
| |
| − | <b>d.</b> Adding 10<sup>-5</sup>[mg/ml] 17-β-estradiol in DMSO to
| |
| − | <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
| |
| − | data-content="UU1250 cloned with hERɑ-Tar chimera">
| |
| − | UERT<i class="entypo-check"></i></button></a>.
| |
| − |
| |
| − | <b>e.</b> Adding 10<sup>-7</sup>[mg/ml] 17-β-estradiol in DMSO to
| |
| − | <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
| |
| − | data-content="UU1250 cloned with hERɑ-Tar chimera">
| |
| − | UERT<i class="entypo-check"></i></button></a>.
| |
| − |
| |
| − |
| |
| − | <b>f.</b> Control - Adding DMSO to
| |
| − | <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
| |
| − | data-content="UU1250 cloned with hERɑ-Tar chimera">
| |
| − | UERT<i class="entypo-check"></i></button></a>.
| |
| − | </p>
| |
| | | | |
| | | | |
