Difference between revisions of "Team:Technion Israel/Modifications/EstoTar"

 
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<br><h4 class="text-center"><b>Introduction</b></h4>
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<br><h4 class="text-center">Introduction</h4>
 
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<br><h4 class="text-center"><b>Design and Implementation</b></h4>
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<br><h4 class="text-center">Design</h4>
 
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<br><h4 class="text-center">Results</h4>
 
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<!-- =========== 1. Intro =========== -->
 
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<h2 class="">Introduction</h2>
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<p class="text-justify">
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<h2 class="">Introduction</h2>
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Our attempts to fuse two segments originating from different organisms to design  
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<p class="text-justify">
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a new receptor was met with great challenges.  These specific segments were the LBD  
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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>
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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
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transduction in response to estrogenic compounds. Despite the fact
 +
that bacterial chemoreceptors are comprised of a two component system and
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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
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estrogen binding to its domain. This led us to design and construct the new
 +
hybrid: hERα-Tar <b> (1)</b>.
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</p>
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</div>
  
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<br>
  
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<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 of hHERa receptor from phyr )<b>
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<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;">
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</a>
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<p class="text-center ocenter"><b>Fig. 1:</b> Predicted structure of human estrogen receptor (hERα) <b>(3)</b> </p>
 
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<!-- =========== 2: conection to the project =========== -->
 
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<h1 class="text-center"><u>Procedure:</u></h1>
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<h2 class="">Design</h2>
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<p class="text-justify">
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<p class="text-justify">
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The <a href="https://2016.igem.org/Team:Technion_Israel/Modifications/Intein">intein-gBlock</a> was designed with the estrogen
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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"  
 
<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>
 
LBD<i class="entypo-check"></i></button></a>
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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"  
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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">
 
data-content="Ligand Binding Domain">
 
LBD<i class="entypo-check"></i></button></a> in the intein gBlock.  
 
LBD<i class="entypo-check"></i></button></a> in the intein gBlock.  
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HAMP<i class="entypo-check"></i></button></a>
 
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.  
 
domain, which is located at the C-terminal end of the last transmembrane segment of Tar, to get a final hybrid product hERα-Tar.  
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The new chimera was cloned to UU1250 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">
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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>
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UERT<i class="entypo-check"></i></button></a>. To the best of our knowledge, this design and cloning has never been reported before.<br>  
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</p>
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<div class="col-md-6 col-md-offset-3 col-sm-12"> <a class="pop ocenter">
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<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;">
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</a>
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<p class="text-center"><b>Fig. 1:</b> The hERɑ-Tar chimera circuit.</p>
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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>  
  
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<br>
 
−
In order to predict the feasibility of this new hybrid, a 3D model was made using the Phyre2 Fold Recognition server (3).
 
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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.<br>
 
 
<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">
 
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. 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">
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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.
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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.
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<!-- ==================== 3: Resuls ==================== -->
 
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<h1 class="text-center"><u>Results</u></h1>
 
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<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  
 
The 3D structure of the hERα-Tar, as can be seen in figure 1, clearly indicates an incorrect folding of the  
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<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) domain, which is located invariably at the C-terminal end of the last transmembrane segment.">
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<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>
 
HAMP<i class="entypo-check"></i></button></a>
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region and thus an overall incorrect structure. Nevertheless, the rest of the tests were conducted in hope for successful results.  
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region, and thus an overall incorrect structure. Nevertheless, the rest of the tests were conducted in hope for successful results.
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<b class="bg-danger">====================**3d modeling of est-tar**====================</b>
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</p><br>
 
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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 2. 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.
 
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<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">
 
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<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>
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<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>
 
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<p class="text-justify">b.</p>
 
<p class="text-justify">b.</p>
 
<a class="pop">
 
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<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>
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<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>
 
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<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>
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<br>
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<br>
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<div class="row">
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<p class="text-justify">
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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>
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</div>
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<br>
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<div class="row">
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<div class="col-md-6 col-sm-12">
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<p class="text-justify">a.</p>
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<a class="pop">
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<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>
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</a>
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</div>
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<div class="col-md-6 col-sm-12">
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<p class="text-justify">b.</p>
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<a class="pop">
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<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>
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</a>
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</div>
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<div class="col-md-12 col-sm-12">
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<p class="text-justify">
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<b>Fig. 2:</b>
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<a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
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data-content="UU1250 cloned with  hERɑ-Tar chimera fused with GFP">
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UERTG<i class="entypo-check"></i></button></a>
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under a fluorescence microscope. <b>a.</b> Under white light. <b>b.</b> Under fluorescence light at 490nm excitation.
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</p>
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<b>Fig. 2:</b>
 
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<a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
 
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data-content="UU1250 cloned with  hERɑ-Tar chimera fused with GFP">
 
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UERTG<i class="entypo-check"></i></button></a>
 
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under a fluorescence microscope. <b>a.</b> Under fluorescence light at 490nm excitation. <b>b.</b> Under white light.
 
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<br>
 
<br>
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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. 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>
+
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>
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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 <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"  
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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">
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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.
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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>
 
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<b class="bg-danger">====================** Video #1 **====================</b>
 
 
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<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">
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</video>
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<p class="text-justify">b.</p>
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<video autoplay loop class="embed-responsive-item img-cont" width="360">
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<source src="https://static.igem.org/mediawiki/2016/e/e8/T--Technion_Israel--est_video_1b.mp4" type="video/mp4">
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</video>
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 +
<p class="text-center">
 
<b>Video 1:</b>  
 
<b>Video 1:</b>  
−
<b class="bg-danger">Link to microscope assay. </b>
+
<a href="https://2016.igem.org/Team:Technion_Israel/Experiments" >microscope assay</a>.
 
<b>a.</b> Adding 10<sup>-5</sup>[mg/ml] 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"  
 
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In attempts to understand and repeat this phenomenon, a range of estradiol concentrations was tested but none of them succeeded.</p>
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In attempts to understand and repeat this phenomenon, a range of estradiol concentrations was tested but none of them succeeded.</p>  
  
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<br>
 
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<br>
 
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<p class="text-justify">
 
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<b class="bg-danger">====================** Video #2 **====================</b>
 
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</p>
 
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<p class="text-justify">
 
−
<b>Video 2:</b> <a href="">Microscope assay.</a><br>
 
−
<b class="bg-danger">Add the link.</b><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">
 
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UERT<i class="entypo-check"></i></button></a>.
 
−
 
−
<b>b.</b> Adding 10<sup>-2</sup>[mg/ml] 17-β-estradiol in DMSO to
 
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<a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
 
−
data-content="UU1250 cloned with  hERɑ-Tar chimera">
 
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UERT<i class="entypo-check"></i></button></a>.
 
−
 
−
<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"
 
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data-content="UU1250 cloned with  hERɑ-Tar chimera">
 
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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">
 
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UERT<i class="entypo-check"></i></button></a>.
 
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−
 
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<b>f.</b> Control - Adding DMSO to
 
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<a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true"
 
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data-content="UU1250 cloned with  hERɑ-Tar chimera">
 
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UERT<i class="entypo-check"></i></button></a>.
 
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</p>
 
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<h2>Outlook</h2>
 
<p class="text-justify">
 
<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
+
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) domain, which is located invariably at the C-terminal end of the last transmembrane segment.">
+
<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>
 
HAMP<i class="entypo-check"></i></button></a>
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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.
+
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 results of the GFP also fit this assumption, as the chimera was unsuccessful at crossing the membrane, probable due to the HAMP.  
+
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, and seems to important in regulating the coiled-coil interactions that seem to be involved in signal propagation, which further supports our assumption. (5,2)<br><br><br>
+
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 fully understand the cause of failure and overcome the obstacles faced in this attempt of generating a new chemoreceptor which reacts to estrogen derivatives.  
+
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 main problems and a way to overcome them are as follows:<br>
−
-The estrogen derivatives used were solubile only in hydrophics solvents which are lethat to the bacteria. The use of estrogen soluble in water or other non-lethal solvents should solve this problem.<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 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 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>
−
-The hERα does not naturally contain a HAMP region. Using a chemoreceptor that does not have a HAMP domain might help overcoming this obstacle. 
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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>
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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>‏
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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>
 
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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>‏
 
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Latest revision as of 01:34, 20 October 2016

S.tar, by iGEM Technion 2016

S.tar, by iGEM Technion 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.

‏





S.tar, by iGEM Technion 2016