Difference between revisions of "Team:Technion Israel/Modifications/EstoTar"
| (133 intermediate revisions by 8 users not shown) | |||
| Line 30: | Line 30: | ||
/* ========== END: and effects ========== */ | /* ========== END: and effects ========== */ | ||
| − | . | + | .references { |
font-size: 16px; | font-size: 16px; | ||
| − | |||
| − | |||
} | } | ||
| Line 60: | Line 58: | ||
position: relative; | position: relative; | ||
} | } | ||
| + | |||
| + | .ocenter { | ||
| + | margin: auto; | ||
| + | } | ||
| + | |||
</style> | </style> | ||
| Line 136: | Line 139: | ||
<!-- =============== Tabs names: =============== --> | <!-- =============== Tabs names: =============== --> | ||
<div class="row"> | <div class="row"> | ||
| − | <div class="col- | + | <div class="col-sm-8 col-sm-offset-2"><!-- 8/12 --> |
| + | |||
<ul class="nav nav-tabs" role="tablist"> | <ul class="nav nav-tabs" role="tablist"> | ||
| Line 142: | 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/d/db/T--Technion_Israel--icon_intro.png" class="img-responsive img-center cont_tabs" width="75" height="75"> | <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" | + | <br><h4 class="text-center">Introduction</h4> |
</a> | </a> | ||
</li> | </li> | ||
| Line 149: | Line 153: | ||
<a href="#Lab" aria-controls="Lab" role="tab" data-toggle="tab"> | <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"> | <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" | + | <br><h4 class="text-center">Design</h4> |
</a> | </a> | ||
</li> | </li> | ||
| Line 156: | Line 160: | ||
<a href="#Results" aria-controls="Results" role="tab" data-toggle="tab"> | <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"> | <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" | + | <br><h4 class="text-center">Results</h4> |
</a> | </a> | ||
</li> | </li> | ||
| Line 163: | Line 167: | ||
<a href="#outlook" aria-controls="outlook" role="tab" data-toggle="tab"> | <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"> | <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"> | + | <br><h4 class="text-center">Outlook</h4> |
</a> | </a> | ||
</li> | </li> | ||
| Line 178: | Line 182: | ||
<!-- =========== 1. Intro =========== --> | <!-- =========== 1. Intro =========== --> | ||
<div role="tabpanel" class="tab-pane fade in active" id="Peshawar"> | <div role="tabpanel" class="tab-pane fade in active" id="Peshawar"> | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
<!-- ========== Content ========== --> | <!-- ========== Content ========== --> | ||
<div class="row"><!-- #1 row --> | <div class="row"><!-- #1 row --> | ||
| − | <div class="col-sm- | + | <div class="col-sm-8 col-sm-offset-2"><!-- 8/12 --> |
| − | + | <!-- 12 text div --> | |
<div class="row"> | <div class="row"> | ||
| − | + | <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 hERα 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>. | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <br> | ||
| − | + | <!-- 12 img div --> | |
| − | + | <div class="row"> | |
| − | + | <div class="col-md-6 col-md-offset-3 col-sm-12"> | |
| − | + | <a class="pop ocenter"> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/5/53/T--Technion_Israel--hERa_receptor_Fig1.png" class="img-responsive img-center img-cont" width="450" style="cursor: pointer;"> | |
| − | + | </a> | |
| + | <p class="text-center ocenter"><b>Fig. 1:</b> Predicted structure of human estrogen receptor (hERα) <b>(3)</b> </p> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | |||
</div> | </div> | ||
</div><!-- END: #1 row --> | </div><!-- END: #1 row --> | ||
</div> | </div> | ||
<!-- =========== END: Intro =========== --> | <!-- =========== END: Intro =========== --> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| Line 223: | Line 228: | ||
<!-- =========== 2: conection to the project =========== --> | <!-- =========== 2: conection to the project =========== --> | ||
<div role="tabpanel" class="tab-pane fade" id="Lab"> | <div role="tabpanel" class="tab-pane fade" id="Lab"> | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| Line 235: | Line 235: | ||
<div class="row"><!-- #1 row --> | <div class="row"><!-- #1 row --> | ||
| − | <div class="col-sm- | + | <div class="col-sm-8 col-sm-offset-2"><!-- 8/12 --> |
| − | <div | + | <!-- 12 text div --> |
| − | + | <div class="row"> | |
| − | + | <div class="col-md-12 col-sm-12"> | |
| − | + | <h2 class="">Design</h2> | |
| − | + | <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"> | data-content="Ligand Binding Domain"> | ||
| − | + | LBD<i class="entypo-check"></i></button></a> | |
| − | + | site as the splicing inducer. The cDNA sequence was the source for 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> 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 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> | |
| − | + | </p> | |
| + | </div> | ||
| + | </div> | ||
| − | + | <!-- 12 img div --> | |
| − | + | <div class="row"> | |
| − | + | <div class="col-md-6 col-md-offset-3 col-sm-12"> <a class="pop ocenter"> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/c/c1/T--Technion_Israel--estrogencircute.png" class="img-responsive img-center ocenter" width="450" style="cursor: pointer;"> | |
| − | < | + | </a> |
| − | + | <p class="text-center"><b>Fig. 1:</b> The hERɑ-Tar chimera circuit.</p> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</div> | </div> | ||
</div> | </div> | ||
| + | |||
| + | <!-- 12 text div --> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-12 col-sm-12"> | ||
| + | <p class="text-justify"> | ||
| + | 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: #1 row --> | ||
</div> | </div> | ||
<!-- =========== END: 2 - conection to the project =========== --> | <!-- =========== END: 2 - conection to the project =========== --> | ||
| − | |||
<!-- ==================== 3: Resuls ==================== --> | <!-- ==================== 3: Resuls ==================== --> | ||
<div role="tabpanel" class="tab-pane fade" id="Results"> | <div role="tabpanel" class="tab-pane fade" id="Results"> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<!-- ========== Content ========== --> | <!-- ========== Content ========== --> | ||
| Line 294: | Line 296: | ||
<div class="row"><!-- #1 row --> | <div class="row"><!-- #1 row --> | ||
| − | <div class="col-sm- | + | <div class="col-sm-8 col-sm-offset-2"><!-- 8/12 --> |
| + | |||
<div class="cont_box"> | <div class="cont_box"> | ||
<div class="row"> | <div class="row"> | ||
<div class="col-sm-12"> | <div class="col-sm-12"> | ||
| − | + | <h2 class="">Results</h2> | |
<p class="text-justify"> | <p class="text-justify"> | ||
| − | + | The 3D structure of the hERα-Tar, as can be seen in figure 1, 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> | </p><br> | ||
| − | |||
| − | |||
| − | |||
<div class="col-md-6 col-sm-12"> | <div class="col-md-6 col-sm-12"> | ||
<p class="text-justify">a.</p> | <p class="text-justify">a.</p> | ||
<a class="pop"> | <a class="pop"> | ||
| − | <img src="https://static.igem.org/mediawiki/2016/ | + | <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> | </a> | ||
</div> | </div> | ||
| − | + | <div class="row"> | |
<div class="col-md-6 col-sm-12"> | <div class="col-md-6 col-sm-12"> | ||
<p class="text-justify">b.</p> | <p class="text-justify">b.</p> | ||
<a class="pop"> | <a class="pop"> | ||
| − | <img src="https://static.igem.org/mediawiki/2016/ | + | <img src="https://static.igem.org/mediawiki/2016/b/b4/T--Technion_Israel--Tar_fig2b.PNG" class="img-responsive img-center img-cont" width="350" style="cursor: pointer;"><br> |
</a> | </a> | ||
</div> | </div> | ||
| + | </div> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-12 col-sm-12"> | ||
| + | <p class="text-justify"> | ||
| + | <b>Fig. 1:</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> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | <br> | ||
| + | <br> | ||
| + | |||
| + | <div class="row"> | ||
| + | <p class="text-justify"> | ||
| + | The results of the fluorescence microscopy were not promising either. 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 (Fig. 2). In other words, in case the chimera is expressed it | ||
| + | probably will not be localized to the membrane, and thus the chemotaxis system will not function correctly. | ||
| + | </p> | ||
| + | </div> | ||
| + | |||
| + | <br> | ||
| + | |||
| + | <div class="row"> | ||
| + | <div class="col-md-6 col-sm-12"> | ||
| + | <p class="text-justify">a.</p> | ||
| + | <a class="pop"> | ||
| + | <img src="https://static.igem.org/mediawiki/2016/9/9c/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-6 col-sm-12"> | ||
| + | <p class="text-justify">b.</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-12 col-sm-12"> | ||
| + | <p class="text-justify"> | ||
| + | <b>Fig. 2:</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 white light. <b>b.</b> Under fluorescence light at 490nm excitation. | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<div class="col-md-12 col-sm-12"> | <div class="col-md-12 col-sm-12"> | ||
<p class="text-justify"> | <p class="text-justify"> | ||
<br> | <br> | ||
| − | + | Despite the discouraging results from the GFP assay we attempted to conduct 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 solvent that was used to dissolve the compound, 17-β-estradiol, was lethal for the bacteria. Since this is a hydrophobic substance, it is only possible to dissolve it in hydrophobic solvents, such as Ethanol or 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"> | data-content="UU1250 cloned with hERɑ-Tar chimera"> | ||
| − | UERT<i class="entypo-check"></i></button></a> strain | + | 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> | ||
| − | |||
| − | |||
</p> | </p> | ||
| + | </div> | ||
| − | <p class="text-justify"> | + | |
| + | |||
| + | <!-- 12 img div --> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-6 col-sm-12"> | ||
| + | <p class="text-justify">a.</p> | ||
| + | <video autoplay loop class="embed-responsive-item img-cont" width="360"> | ||
| + | <source src="https://static.igem.org/mediawiki/2016/8/8e/T--Technion_Israel--est_video_1a.mp4" type="video/mp4"> | ||
| + | </video> | ||
| + | </div> | ||
| + | |||
| + | <div class="col-md-6 col-sm-12"> | ||
| + | <p class="text-justify">b.</p> | ||
| + | <video autoplay loop class="embed-responsive-item img-cont" width="360"> | ||
| + | <source src="https://static.igem.org/mediawiki/2016/e/e8/T--Technion_Israel--est_video_1b.mp4" type="video/mp4"> | ||
| + | </video> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | <p class="text-center"> | ||
<b>Video 1:</b> | <b>Video 1:</b> | ||
| − | < | + | <a href="https://2016.igem.org/Team:Technion_Israel/Experiments" >microscope assay</a>. |
| − | <b>a.</b> Adding 17-β-estradiol in DMSO to | + | <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" | <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true" | ||
data-content="UU1250 cloned with hERɑ-Tar chimera"> | data-content="UU1250 cloned with hERɑ-Tar chimera"> | ||
| Line 367: | Line 418: | ||
</p> | </p> | ||
| − | + | ||
| − | + | ||
| − | + | ||
<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> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
| Line 418: | Line 430: | ||
</div><!-- END: #1 row --> | </div><!-- END: #1 row --> | ||
</div> | </div> | ||
| + | |||
<!-- ====================END: 3 Resuls ==================== --> | <!-- ====================END: 3 Resuls ==================== --> | ||
| Line 425: | Line 438: | ||
<!-- ==================== 4: Outlook ==================== --> | <!-- ==================== 4: Outlook ==================== --> | ||
<div role="tabpanel" class="tab-pane fade" id="outlook"> | <div role="tabpanel" class="tab-pane fade" id="outlook"> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<!-- ========== Content ========== --> | <!-- ========== Content ========== --> | ||
<div class="row"><!-- #1 row --> | <div class="row"><!-- #1 row --> | ||
| − | <div class="col-sm- | + | <div class="col-sm-8 col-sm-offset-2"><!-- 8/12 --> |
| + | |||
<div class="cont_box"> | <div class="cont_box"> | ||
<div class="row"> | <div class="row"> | ||
<div class="col-sm-12"> | <div class="col-sm-12"> | ||
| − | + | <h2>Outlook</h2> | |
<p class="text-justify"> | <p class="text-justify"> | ||
| − | + | Although the concept of this idea seemed promising, 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 Tar's 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 for the chimera failure, and our results support this assumption. For example, in the modeled 3D structure, it is clear that HAMP region is problematic, as it is clear the HAMP misplaced. | |
| + | Furthermore, the GFP results also fit this assumption, as the chimera did not localize to the membrane, probable due to the HAMP. | ||
| + | Lastly, most of the reported chemotaxis receptors naturally contain a HAMP domain. According to the literature, that area is important for regulating the coiled-coil interactions mediating the signal propagation, which further supports our assumption. <b>(2,5)</b>.<br><br><br> | ||
| + | |||
| + | To conclude, further research is needed in order to 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 soluble only in hydrophobic solvents, which are lethal to bacteria. The use of hydrophilic estrogen derivative or other non- lethal solvents should solve this problem.<br> | ||
| + | * The 3D model indicated of the incorrect folding of the newly designed receptor. A new design focused more on the Tar might aid in solving this problem.<br> | ||
| + | |||
</p> | </p> | ||
</div> | </div> | ||
| Line 458: | Line 473: | ||
</div><!-- ============ END: Tabs ============ --> | </div><!-- ============ END: Tabs ============ --> | ||
| + | |||
| + | <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"target="_blank">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> | ||
| Line 479: | Line 509: | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<br> | <br> | ||
| Line 505: | Line 516: | ||
<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> | ||
| − | + | </div> | |
</body> | </body> | ||
</html> | </html> | ||
{{:Team:Technion_Israel/supporters}} | {{:Team:Technion_Israel/supporters}} | ||
Latest revision as of 01:34, 20 October 2016
Introduction
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.
hERɑ 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 hERα 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 (1).
Fig. 1: Predicted structure of human estrogen receptor (hERα) (3)
Design
The intein-gBlock was designed with the estrogen
LBD
site as the splicing inducer. The cDNA sequence was the source for the
LBD in the intein gBlock.
This design provided the team an opportunity to easily extract the
LBD and fuse it to
HAMP
domain, which is located at the C-terminal end of the last transmembrane segment of Tar, to get a final hybrid product hERα-Tar.
The new chimera was cloned to UU1250 to generate the new strain: UERT. To the best of our knowledge, this design and cloning has never been reported before.
Fig. 1: The hERɑ-Tar chimera circuit.
In order to predict the feasibility of this new hybrid, a 3D model was made using the Phyre2 Fold Recognition server (3).
Later, in order to confirm the correct localization at both poles of the bacterial membrane (4), a GFP reporter protein was fused to the hERα-Tar chimera and tested with fluorescence microscopy.
Finally, a “Chip Microscope assay” was conducted to study the effects of 17- β-estradiol on the chemotaxis system of the
UERT strain. In short, a suspension of the
UERT 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.
Results
The 3D structure of the hERα-Tar, as can be seen in figure 1, clearly indicates an incorrect folding of the HAMP region, and thus an overall incorrect structure. Nevertheless, the rest of the tests were conducted in hope for successful results.
a.
b.
Fig. 1: a. a model demonstration of a predict structure of hERɑ-Tar chimera. b. a model demonstration of a predict structure of Tar chemoreceptor.
The results of the fluorescence microscopy were not promising either. 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 (Fig. 2). In other words, in case the chimera is expressed it probably will not be localized to the membrane, and thus the chemotaxis system will not function correctly.
a.
b.
Fig. 2: UERTG under a fluorescence microscope. a. Under white light. b. Under fluorescence light at 490nm excitation.
Despite the discouraging results from the GFP assay we attempted to conduct 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.
Later, we concluded that the solvent that was used to dissolve the compound, 17-β-estradiol, was lethal for the bacteria. Since this is a hydrophobic substance, it is only possible to dissolve it in hydrophobic solvents, such as Ethanol or 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.
A noteworthy phenomenon that occurred during one of the microscope tests, was when a solution of 17-β-estradiol dissolved in DMSO (concentration 10-5 mg/ml) was added to the
UERT 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.
a.
b.
Video 1: microscope assay. a. Adding 10-5[mg/ml] 17-β-estradiol in DMSO to UERT. b. Control - Adding DMSO to UERT.
In attempts to understand and repeat this phenomenon, a range of estradiol concentrations was tested but none of them succeeded.
Outlook
Although the concept of this idea seemed promising, the PctA-Tar and NarX chimeras had more potential to succeed, due to their structure similarity to the native Tar chemoreceptor (they all contain
HAMP
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 Tar's 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 for the chimera failure, and our results support this assumption. For example, in the modeled 3D structure, it is clear that HAMP region is problematic, as it is clear the HAMP misplaced.
Furthermore, the GFP results also fit this assumption, as the chimera did not localize to the membrane, probable due to the HAMP.
Lastly, most of the reported chemotaxis receptors naturally contain a HAMP domain. According to the literature, that area is important for regulating the coiled-coil interactions mediating the signal propagation, which further supports our assumption. (2,5).
To conclude, further research is needed in order to 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:
* The estrogen derivatives used were soluble only in hydrophobic solvents, which are lethal to bacteria. The use of hydrophilic estrogen derivative or other non- lethal solvents should solve this problem.
* The 3D model indicated of the incorrect folding of the newly designed receptor. A new design focused more on the Tar might aid in solving this problem.
References:
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.
2. HULKO, Michael, et al. The HAMP domain structure implies helix rotation in transmembrane signaling. Cell, 2006, 126.5: 929-940.
3. Phyre2 modeling server.
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.
5. WADHAMS, George H.; ARMITAGE, Judith P. Making sense of it all: bacterial chemotaxis. Nature Reviews Molecular Cell Biology, 2004, 5.12: 1024-1037.
