Difference between revisions of "Team:Technion Israel/Proof"
| (44 intermediate revisions by 7 users not shown) | |||
| Line 18: | Line 18: | ||
.desk_wrapper { | .desk_wrapper { | ||
position: relative; | position: relative; | ||
| − | + | background: white; /* For browsers that do not support gradients */ | |
background: -webkit-linear-gradient(white, #ecf7fb, white); /* For Safari 5.1 to 6.0 */ | background: -webkit-linear-gradient(white, #ecf7fb, white); /* For Safari 5.1 to 6.0 */ | ||
background: -o-linear-gradient(white, #ecf7fb, white); /* For Opera 11.1 to 12.0 */ | background: -o-linear-gradient(white, #ecf7fb, white); /* For Opera 11.1 to 12.0 */ | ||
| Line 48: | Line 48: | ||
Every in-content-page img needs to have this class of col. | Every in-content-page img needs to have this class of col. | ||
*/ | */ | ||
| − | . | + | .referances { |
font-size: 16px; | font-size: 16px; | ||
padding: 20px; | padding: 20px; | ||
| Line 61: | Line 61: | ||
margin: 0 auto; | margin: 0 auto; | ||
} | } | ||
| − | + | ||
| − | . | + | .vcenter { |
| − | + | display: inline-block; | |
| + | vertical-align: middle; | ||
| + | float: none; | ||
} | } | ||
| − | |||
</style> | </style> | ||
| Line 126: | Line 127: | ||
<div class="row"> | <div class="row"> | ||
<div class="col-xs-12"> | <div class="col-xs-12"> | ||
| − | <img src="https://static.igem.org/mediawiki/2016/4/49/T--Technion_Israel--Proofofconceptcov.jpg" class="img-responsive img-center" width="100%"> | + | <img src="https://static.igem.org/mediawiki/2016/4/49/T--Technion_Israel--Proofofconceptcov.jpg" class="img-responsive img-center cont_cover" width="100%"> |
</div> | </div> | ||
</div> | </div> | ||
| Line 132: | Line 133: | ||
| − | + | <!-- =========== Content =========== --> | |
| − | + | <div class="row"><!--row --> | |
| − | + | <div class="col-sm-8 col-sm-offset-2"><!-- 10/12 --> | |
| − | + | <!-- ============ Tabs: ============ --> | |
| − | + | ||
| − | + | ||
| − | |||
| − | + | <!-- =============== Tabs names: =============== --> | |
| − | + | <div class="row"> | |
| − | + | <div class="col-md-12"> | |
| − | + | ||
| − | + | <ul class="nav nav-tabs" role="tablist"> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | <li role="presentation" class="col-sm-6 col-xs-6"> | |
| − | + | <a href="#111" aria-controls="111" role="tab" data-toggle="tab"> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/7/78/T--Technion_Israel--narXicon_%282%29.png" class="img-responsive img-center cont_tabs" width="75" height="75"> | |
| − | + | <br><h4 class="text-center">PctA-Tar</h4> | |
| − | + | </a> | |
| − | + | </li> | |
| − | + | ||
| + | <li role="presentation" class="col-sm-6 col-xs-6"> | ||
| + | <a href="#222" aria-controls="222" role="tab" data-toggle="tab"> | ||
| + | <img src="https://static.igem.org/mediawiki/2016/9/9c/T--Technion_Israel--histiminicon.png" class="img-responsive img-center cont_tabs" width="75" height="75"> | ||
| + | <br><h4 class="text-center">Histamine-Tar</h4> | ||
| + | </a> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </div> | ||
| + | </div> | ||
| + | <!-- ========== End: Tabs panel ========== --> | ||
| + | |||
| + | |||
| + | |||
| + | <!--=======================================================================================================--> | ||
| + | |||
| + | |||
| + | |||
| + | <!-- =========== Tabs' content =========== --> | ||
| + | <div class="tab-content"> | ||
| + | |||
| + | |||
| + | <!-- 111111111111111111111111111111111111111111111111111111111 --> | ||
| + | <div role="tabpanel" class="tab-pane fade in active" id="111"> | ||
| + | <div class="cont_box"> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | <div class="row"> | ||
| + | <div class="col-md-12 col-sm-12"> | ||
| + | <h2>PctA-Tar chimera Introduction</h2> | ||
</div> | </div> | ||
| − | + | </div> | |
| + | <br> | ||
| + | <br> | ||
| − | + | <!--6 text - 6 img div--> | |
| − | <div class=" | + | <div class="row"> |
| − | < | + | <div class="col-md-6 col-sm-12 vcenter"><!--6 text--> |
| + | <p class="text-justify"> | ||
| + | One of S.Tars sub-projects focused on altering the LBD of Tar chemoreceptor in order to design new hybrid chimeras. That was accomplished by replacing the LBD of the original Tar chemoreceptor with a new one, from a different source, without modifying Tar signaling region. As a proof of concept for the newly designed Tar chimeras and the S.Tar project, we focused on testing the PctA-Tar hybrid. | ||
| + | </p> | ||
| + | </div><!-- | ||
| + | --><div class="col-md-6 col-sm-12 vcenter"><!--6 img div--> | ||
| + | <a class="pop ocenter"> | ||
| + | <img src="https://static.igem.org/mediawiki/2016/thumb/7/7d/T--Technion_Israel--Tar_pctA.png/800px-T--Technion_Israel--Tar_pctA.png" class="img-responsive img-center img-cont" width="450"> | ||
| + | </a> | ||
| + | <p class="text-center"><b>Fig. 1:</b> Scheme of native Tar chemoreceptor, native PctA receptor and PctA-Tar chimera. Adapted from <b> (1) </b></p> | ||
| + | </div> | ||
| + | </div> | ||
| − | + | <br> | |
| − | + | <br> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | <div class="row"> | |
| − | + | <div class="col-md-12 col-sm-12"> | |
| − | < | + | <div class="col-md-12 col-sm-12"> |
| − | + | <p class="text-justify"> | |
| − | + | PctA is a chemoreceptor found in the <i> Pseudomonas Aeruginosa </i>.It mediates | |
| − | + | chemotaxis towards amino acids and away from organic compounds. It can sense all | |
| + | amino acids except for Aspartate <b>(1)</b>. | ||
| + | <br>To construct this chimera, the LBD sequence of the PctA was obtained from the <a href="http://www.pseudomonas.com/"target="_blank"><i> Pseudomonas </i> genome database</a>, | ||
| + | while the signaling region of Tar was obtained from the iGEM parts catalog | ||
| + | <a href="http://parts.igem.org/Part:BBa_K777000" target="_blank">(K777000)</a>. Using these two sequences, we built a Biobrick part <a href="http://parts.igem.org/Part:BBa_K1992007" target="_blank">(K1992007)</a> | ||
| + | which was tested by transforming the device to bacteria that lacks chemoreceptors - <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true" | ||
| + | data-content="An E.coli derivative, which lacks chemoreceptors genes, means this strain does not obtain chemotaxis ability (Parkinson J S, University of Utah)."> | ||
| + | UU1250<i class="entypo-check"></i></button></a>. It is important | ||
| + | to note that this chimera has been constructed before in the literature <b>(1)</b>. | ||
| + | </p> | ||
| + | </div> | ||
| − | </ | + | <div class="col-sm-6 col-sm-offset-3"> |
| + | <a class="pop ocenter"> | ||
| + | <img src="https://static.igem.org/mediawiki/2016/thumb/3/30/T--Technion_Israel--pctacircute.png/800px-T--Technion_Israel--pctacircute.png" class="img-responsive img-center" width="500" style="cursor: pointer;"> | ||
| + | </a> | ||
| + | <p><b>Fig. 2:</b> Biobrick device of the PctA-Tar chimera.</p> | ||
</div> | </div> | ||
| + | |||
</div> | </div> | ||
| − | + | </div> | |
| − | |||
| − | |||
| − | |||
| − | + | <div class="row"> | |
| − | + | <div class="col-md-12 col-sm-12"> | |
| − | + | <h2>Test and results</h2> | |
| − | + | </div> | |
| − | + | </div> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <div class="col-sm-8 col-sm-offset-2"><!-- 8/12 --> | + | <div class="row"> |
| − | + | <div class="col-md-12 col-sm-12"> | |
| − | + | <p class="text-justify"> | |
| − | + | As an initial step, we generated a 3D model of the PctA-Tar chimera, figure 3, using the | |
| − | + | Phyre2 Protein Fold Recognition server to assure the correct folding of both the LBD and the signaling regions. | |
| − | + | </p> | |
| − | + | </div> | |
| − | + | </div> | |
| − | + | ||
| − | + | <br> | |
| + | |||
| + | <div class="row"> | ||
| + | <div class="col-sm-8 col-sm-offset-2"><!-- 8/12 --> | ||
| + | <a class="pop ocenter"> | ||
| + | <img src="https://static.igem.org/mediawiki/2016/6/62/T--Technion_Israel--Proof_fig3.png" class="img-responsive img-center img-cont" width="700" style="cursor: pointer;"> | ||
| + | </a> | ||
| + | <p><b>Fig. 3: </b> PctA-Tar chimera 3D structure. The Tar signaling regions is in gray, the PctA LBD is in red.</p> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | <br> | ||
| + | <br> | ||
| + | |||
| + | <!--6 text - 6 img div--> | ||
<div class="row"> | <div class="row"> | ||
<div class="col-md-6 col-sm-12 vcenter"><!--6 text--> | <div class="col-md-6 col-sm-12 vcenter"><!--6 text--> | ||
<p class="text-justify"> | <p class="text-justify"> | ||
| − | Following | + | Following transformation, a swarming plate assay was performed in order to |
confirm the functionality of the hybrid receptor. A scheme of the assay is | confirm the functionality of the hybrid receptor. A scheme of the assay is | ||
| − | presented below | + | presented below (figure 4). It is important to mention that this assay was |
performed on BA medium as the original assay on TB medium failed. From the | performed on BA medium as the original assay on TB medium failed. From the | ||
| − | results seen | + | results seen in figure 5, it is |
| − | + | clear that the chimera functions and controls the chemotactic ability of | |
| − | the bacteria | + | the bacteria leading to swarming response. |
| + | This is compared to the control, UU1250 strain, that did not show any chemotactic ability as expected and no chemotactic rings were formed. | ||
| + | |||
</p> | </p> | ||
</div><!-- | </div><!-- | ||
| Line 250: | Line 296: | ||
</div> | </div> | ||
</div> | </div> | ||
| + | |||
| + | <br> | ||
| + | <br> | ||
<br> | <br> | ||
| − | |||
| − | |||
| − | + | <div class="row"> | |
| − | + | <div class="col-md-4 col-sm-12"> | |
| − | + | <p class="text-justify">a.</p> | |
| − | + | <a class="pop"> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/thumb/5/52/T--Technion_Israel--pcta_figure1a.JPG/600px-T--Technion_Israel--pcta_figure1a.JPG" class="img-responsive img-center img-cont" width="200" style="cursor: pointer;"><br> | |
| − | + | </a> | |
| − | + | </div> | |
| − | + | <div class="col-md-4 col-sm-12"> | |
| − | + | <p class="text-justify">b.</p> | |
| − | + | <a class="pop"> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/thumb/6/6a/T--Technion_Israel--pcta_figure1b.JPG/600px-T--Technion_Israel--pcta_figure1b.JPG" class="img-responsive img-center img-cont" width="200" style="cursor: pointer;"><br> | |
| − | + | </a> | |
| − | + | </div> | |
| − | + | <div class="col-md-4 col-sm-12"> | |
| − | + | <p class="text-justify">c.</p> | |
| − | + | <a class="pop"> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/thumb/d/d2/T--Technion_Israel--pcta_figure1c.JPG/600px-T--Technion_Israel--pcta_figure1c.JPG" class="img-responsive img-center img-cont" width="200" style="cursor: pointer;"><br> | |
| − | + | </a> | |
| − | + | </div> | |
| − | + | <div class="col-md-12 col-sm-12"> | |
| − | + | <p class="text-justify"> | |
| − | + | <b>Fig. 5:</b> Swarming assay for attractant response of the PctA-Tar chimera. | |
| − | + | <b>a.</b> PctA chimera, <b>b.</b> Negative control- UU1250 strain w/o the Tar expression plasmid, <b>c. </b> positive control - ΔZras strain expressing all chemoreceptors. | |
| − | + | <br><br><br> | |
| − | + | </p> | |
| − | + | </div> | |
| − | + | </div> | |
| − | + | <div class="row"> | |
| − | + | <div class="col-md-12 col-sm-12 vcenter"> | |
| − | + | <p class="text-justify"> | |
| − | + | Next, to prove that the chimera is localized in the membrane, GFP was fused | |
| − | + | to its C-terminus with a short linker sequence <a href="http://parts.igem.org/Part:BBa_K1992010" target="_blank">(K1992010)</a>, | |
| − | + | figure 6. The results seen in figure 7, show that indeed the chimera is localized to the membrane (poles). | |
| − | + | </p> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</div> | </div> | ||
| − | + | <div class="col-md-6 col-md-offset-3 col-sm-12 vcenter"> | |
| − | < | + | <a class="pop ocenter"> |
| + | <img src="https://static.igem.org/mediawiki/2016/thumb/6/6e/T--Technion_Israel--pctaGFPcircute.png/800px-T--Technion_Israel--pctaGFPcircute.png" class="img-responsive img-center " width="500" style="cursor: pointer;"> | ||
| + | </a> | ||
| + | <p><b>Fig. 6:</b> Biobrick device of the PctA-Tar chimera fused to GFP.</p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <br> | ||
| + | <br> | ||
| + | <br> | ||
| − | < | + | <div class="row"> |
| − | + | <div class="col-sm-8 col-sm-offset-2"> | |
| − | + | <a class="pop ocenter"> | |
| + | <img src="https://static.igem.org/mediawiki/2016/thumb/6/6a/T--Technion_Israel--Tar_pctA_flourecent.png/800px-T--Technion_Israel--Tar_pctA_flourecent.png" class="img-responsive img-center img-cont" width="700" style="cursor: pointer;"> | ||
| + | </a> | ||
| + | <p><b>Fig. 7:</b> Results of GFP fusion. <b>(A)</b> Positive control- <i>E.Coli</i> strain expressing GFP protein,<b>(B)</b> | ||
| + | Negative control- UU1250 strain expressing Tar chemoreceptor, <b>(C)</b> UU1250 strain expressing Tar-GFP chemoreceptor, <b>(D)</b> | ||
| + | UU1250 strain expressing PctA-Tar-GFP Chimera, fluorescence (490nm excitation). | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | <br> | ||
| + | <br> | ||
| + | <br> | ||
| − | + | <div class="row"> | |
| − | + | <div class="col-md-12 col-sm-12"> | |
| − | + | <p class="text-justify"> | |
| − | + | Finally, in the video below is a working concept of the <a href="https://2016.igem.org/Team:Technion_Israel/Design">FlashLab project</a> - a chip that serves | |
| − | + | as a detection tool based on the chemotaxis system of <I>E. coli</I> bacteria. In the video, a commercial ibidi | |
| − | + | microfluidic chip filled with a suspension of bacteria expressing the chimera and chromoprotein | |
| − | + | (<a href="http://parts.igem.org/Part:BBa_J23100" target="_blank">J23100</a> + <a href="http://parts.igem.org/Part:BBa_K1357009" target="_blank">K1357009</a>) can been seen. | |
| − | + | A solution of repellent (10<sup>-3</sup>M Tetrachloroethylene) was added to the chip | |
| − | + | and the displacement of the bacteria was monitored and recorded. | |
| − | + | </p> | |
| − | + | </div> | |
| − | + | </div> | |
| + | |||
| + | <br> | ||
| − | + | <div class="row"> | |
| − | + | <div class="col-md-12 col-sm-12"> | |
| − | + | <br> | |
| − | + | <a class="pop ocenter"> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/thumb/9/95/T--Technion_Israel--FlashLab.png/800px-T--Technion_Israel--FlashLab.png" class="img-responsive img-center img-cont" style="cursor: pointer;"><br> </a> | |
| − | + | <p class="text-center"> | |
| − | + | <b>Fig. 8:</b> A steps scheme of the <a href="https://2016.igem.org/Team:Technion_Israel/Design">FlashLab</a> concept: Add bacteria expressing both the chemoreceptor of your choice and a chromoprotein to a fluidic chip. Add the tested sample to the chip. If the chemoreceptor detecets the substance in the sample, a displacement of the bacteria will become visible. | |
| − | + | .</p> | |
| − | + | </div> | |
| − | + | </div> | |
| − | + | ||
| − | + | <br> | |
| + | <br> | ||
| + | <br> | ||
| + | |||
| + | <!--6 text - 6 img div--> | ||
<div class="row"> | <div class="row"> | ||
<div class="col-md-6 col-sm-12 vcenter"><!--6 text--> | <div class="col-md-6 col-sm-12 vcenter"><!--6 text--> | ||
| − | |||
<p class="text-justify"> | <p class="text-justify"> | ||
| − | + | In video 1, the displacement of the bacteria can be clearly seen in test chip compared to the control chip. | |
| − | + | ||
| − | + | ||
</p> | </p> | ||
</div><!-- | </div><!-- | ||
| Line 357: | Line 410: | ||
</a> | </a> | ||
<br> | <br> | ||
| − | |||
| − | |||
| − | |||
<p class="text-center"><b>Video 1:</b> from left to right: | <p class="text-center"><b>Video 1:</b> from left to right: | ||
(1) PctA-Tar chimera with Tetrachloroethylene repellent added. | (1) PctA-Tar chimera with Tetrachloroethylene repellent added. | ||
| − | (2) PctA-Tar chimera with | + | (2) PctA-Tar chimera with Motility buffer added (control). |
</p> | </p> | ||
</div> | </div> | ||
| − | |||
</div> | </div> | ||
| − | + | <br><br><br> | |
| + | <!-- 12 text div --> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-12 col-sm-12"> | ||
| + | <p class="text-justify"> | ||
| + | With the supporting evidence of the results presented above, it can be concluded | ||
| + | that both concepts: LBD altering and the <a href="https://2016.igem.org/Team:Technion_Israel/Design">FlashLab</a> platform, have been proven and work under real life conditions and seem promising for detection of various substances in the near future. | ||
| + | |||
| + | </p> | ||
| + | </div> | ||
</div> | </div> | ||
| + | |||
| + | </div> | ||
| + | </div> | ||
| + | <!--===============--> | ||
| + | <!-- ======================== END: 111 ======================== --> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | <!-- 222222222222222222222222222222222222222222222222222222222 --> | ||
| + | <div role="tabpanel" class="tab-pane fade" id="222"> | ||
| + | <div class="cont_box"> | ||
| + | |||
| + | <!-- Mini headline --> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-12 col-sm-12"> | ||
| + | <h2>Histamine-Tar Introduction</h2> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | <br> | ||
| + | |||
| + | <!--6 text - 6 img div--> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-6 col-sm-12 vcenter"><!--6 text--> | ||
| + | <p class="text-justify"> | ||
| + | The heart of the S.Tar project is the Tar chemoreceptor, one of four <i>E. coli</i> receptors. | ||
| + | Our goal is to create an engineered bacteria which has chemotaxis receptors sensitive | ||
| + | to materials other than its native ligands. We show that <i>E. | ||
| + | coli</i> can be engineered to respond to completely new materials. By changing Tar’s ligand binding domain | ||
| + | (LBD) to other LBDs from various sources or by mutating it. | ||
| + | </p> | ||
| + | </div><!-- | ||
| + | --><div class="col-md-6 col-sm-12 vcenter"><!--6 img div--> | ||
| + | <a class="pop ocenter"> | ||
| + | <img src="https://static.igem.org/mediawiki/2016/7/72/T--Technion_Israel--His_seq.png" class="img-responsive img-center img-cont" width="450"> | ||
| + | </a> | ||
| + | <p class="text-center"><b>Fig. 1:</b> Sequencing Results. "Query" describes the native Tar LBD | ||
| + | sequence and "Sbjct" describes the design mutations sequence. The mutation region is marked with color (blue or red).</p> | ||
</div> | </div> | ||
| + | </div> | ||
| + | |||
| + | <br> | ||
| + | <br> | ||
| + | |||
| + | <!-- 12 text div --> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-12 col-sm-12"> | ||
| + | <p class="text-justify"> | ||
| + | The bacterial world offers a relatively small selection of chemoreceptors in comparison to | ||
| + | the vast number of possible ligands. These receptors evolved specifically to recognize substances | ||
| + | which benefit or harm the organism. On top of that the fact that the majority of known | ||
| + | receptors today are not well characterized meant that we had very few options of creating chimeric | ||
| + | receptors as we initially planned.<br><br> | ||
| + | In light of the above we had to turn to a new path – redesigning the Tar chemoreceptor to bind a | ||
| + | different ligand using computational biology - The Rosetta <a href="https://2016.igem.org/Team:Technion_Israel/Software">software</a>. | ||
| + | Out of the Rosetta’s 870 suggested mutations only 11 variants were eventually cloned into the native Tar ligand-binding domain (LBD). | ||
| + | See <a href="https://2016.igem.org/Team:Technion_Israel/Modifications/Rosetta">Computational Design</a> page for more information regarding the design process. | ||
| + | Out of all the tested variants, only one was discovered to be attracted to Histamine. Sequencing results | ||
| + | showed that the only mutations to occur in this variant were those planned by the Rosetta’s design. The desired sequences can be seen in figure 1. | ||
| + | </p> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | ||
| − | + | ||
| − | < | + | <div class="row"> |
| − | <div class=" | + | <div class="col-sm-6 col-sm-offset-3"> |
| − | + | <a class="pop ocenter"> | |
| + | <img src="https://static.igem.org/mediawiki/2016/thumb/5/5c/T--Technion_Israel--Scheme_His.png/510px-T--Technion_Israel--Scheme_His.png" class="img-responsive img-center img-cont" width="500" style="cursor: pointer;"> | ||
| + | </a> | ||
| + | <p><b>Fig. 2:</b> Histamine-Tar filtering process scheme</p> | ||
| + | </div> </div> | ||
| + | |||
| + | |||
| + | |||
| + | <div class="row"> | ||
| + | <div class="col-md-12 col-sm-12"> | ||
| + | <h2>Test and results</h2> | ||
| + | </div> | ||
| + | |||
| + | <div class="row"> | ||
| + | <div class="col-md-12 col-sm-12"> | ||
| + | <p class="text-justify"> | ||
| + | We observed the bacteria’s response to the attractant, | ||
| + | Histamine, by using a microscope. It is evident in figure 3b that roughly 20 minutes after the addition of the | ||
| + | Histamine, the bacteria concentration in the vicinity of the Histamine is much greater | ||
| + | than in the the beginning of the experiment (figure 3a). | ||
| + | |||
| + | </p> | ||
| + | </div> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | + | <!-- 12 img div --> | |
| − | + | <div class="row"> | |
| − | + | <div class="col-sm-8 col-sm-offset-2"><!-- 8/12 --> | |
| − | + | <div class="row"> | |
| − | + | <div class="col-md-6 col-xs-12"> | |
| − | + | <p class="text-justify">a.</p> | |
| − | + | <a class="pop"> | |
| − | </ | + | <img src="https://static.igem.org/mediawiki/2016/9/96/T--Technion_Israel--His0min.png" |
| + | class="img-responsive img-center img-cont" width="220" style="cursor: pointer;"><br> | ||
| + | </a> | ||
| + | </div> | ||
| + | |||
| + | <div class="col-md-6 col-xs-12"> | ||
| + | <p class="text-justify">b.</p> | ||
| + | <a class="pop"> | ||
| + | <img src="https://static.igem.org/mediawiki/2016/5/5f/T--Technion_Israel--His20min.png" class="img-responsive img-center img-cont" width="220" style="cursor: pointer;"><br> | ||
| + | </a> | ||
</div> | </div> | ||
| − | + | ||
| − | + | <div class="col-md-6 col-xs-12"> | |
| − | + | <p class="text-justify">c.</p> | |
| − | + | <a class="pop"> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/9/94/T--Technion_Israel--His_control0min.png" class="img-responsive img-center img-cont" width="220" style="cursor: pointer;"><br> | |
| − | + | </a> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | </ | + | |
</div> | </div> | ||
| − | + | ||
| − | <div class="col-md- | + | <div class="col-md-6 col-xs-12"> |
| − | + | <p class="text-justify">d.</p> | |
| + | <a class="pop"> | ||
| + | <img src="https://static.igem.org/mediawiki/2016/2/25/T--Technion_Israel--His_control20min.png" class="img-responsive img-center img-cont" width="220" style="cursor: pointer;"><br> | ||
| + | </a> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | </div> | + | |
| − | + | <div class="row"> | |
| − | <div class="col-md-12 col-sm-12"> | + | <div class="col-md-12 col-sm-12"> |
| − | + | <p class="text-center"><b>Fig. 3:</b> Microscope results of chemotaxis activity for variant His_9 with 10mM Histamine. (a) Tar-Histamine: before adding Histamine. (b) Tar-Histamine: 20 minutes after adding Histamine. (c) Tar-Histamine: before adding the Motility buffer (control solution). (d) Tar-Histamine: 20 minutes after adding the Motility buffer. | |
| − | + | </p> | |
| + | </div> | ||
</div> | </div> | ||
| − | |||
| + | </div> | ||
| + | </div> | ||
| − | |||
<br> | <br> | ||
<br> | <br> | ||
| Line 476: | Line 571: | ||
<div class="col-md-12 col-sm-12"> | <div class="col-md-12 col-sm-12"> | ||
<p class="text-justify"> | <p class="text-justify"> | ||
| − | To prove the correct localization of the | + | To prove the correct localization of the Tar-Histamine, GFP was fused to its C-terminus with a short linker sequence <a href="http://parts.igem.org/Part:BBa_E0040" target="_blank">(E0040)</a> . The results seen in figure 4 show that indeed the chimera is localized to the membrane (poles). |
| Line 486: | Line 581: | ||
<br> | <br> | ||
<br> | <br> | ||
| − | |||
| − | |||
<!-- 12 img div --> | <!-- 12 img div --> | ||
<div class="row"> | <div class="row"> | ||
| − | + | <div class="col-sm-8 col-sm-offset-2"><!-- 8/12 --> | |
| − | + | <a class="pop ocenter"> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/thumb/e/e3/T--Technion_Israel--His-GFP.png/800px-T--Technion_Israel--His-GFP.png" class="img-responsive img-center img-cont" width="600" style="cursor: pointer;"> | |
| − | + | </a> | |
| − | + | <br> | |
| − | + | <p class="text-center"> | |
| − | + | <b>Fig. 4:</b> Results of GFP fusion. (A) Positive control- E.Coli strain expressing GFP protein, (B) Negative control- UU1250 strain expressing Tar chemoreceptor, (C) UU1250 strain expressing Tar-GFP chemoreceptor, (D) UU1250 strain expressing Histamine-Tar-GFP Chimera, fluorescence (490nm excitation). | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | < | + | |
| − | < | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | </p> | |
| − | + | ||
| − | + | ||
</div> | </div> | ||
| − | </div> | + | </div> |
| − | + | ||
| − | + | ||
<br> | <br> | ||
<br> | <br> | ||
| Line 532: | Line 604: | ||
<div class="col-md-6 col-sm-12 vcenter"><!--6 text--> | <div class="col-md-6 col-sm-12 vcenter"><!--6 text--> | ||
<p class="text-justify"> | <p class="text-justify"> | ||
| − | Finally, | + | Finally, in the video below, a working concept of the <a href="https://2016.igem.org/Team:Technion_Israel/Design">FlashLab project</a> - |
| − | a chip that serves as a detection tool based on the chemotaxis system of E. | + | a chip that serves as a detection tool based on the chemotaxis system of <i>E. |
| − | coli bacteria | + | coli</i> bacteria is presented. In the video, a commercial ibidi microfluidic chip filled with a suspension of |
| − | bacteria expressing the chemoreceptor and chromoprotein | + | bacteria expressing both the chemoreceptor and chromoprotein |
| − | <a href="http://parts.igem.org/Part:BBa_K1357008" target="_blank"> | + | (<a href="http://parts.igem.org/Part:BBa_K1992011" target="_blank">K1992011</a> + <a href="http://parts.igem.org/Part:BBa_K1357008" target="_blank">K1357008</a>) can been seen. |
| − | A solution of | + | A solution of attractant (10<sup>-3</sup>M Histamine) |
| − | was added to the chip and | + | was added to the chip and bacteria displacement was monitored and recorded |
| − | + | ||
</p> | </p> | ||
</div><!-- | </div><!-- | ||
| Line 551: | Line 622: | ||
</video--> | </video--> | ||
<p class="text-center"><b>Video 1:</b> from left to right: | <p class="text-center"><b>Video 1:</b> from left to right: | ||
| − | (1) Histamine-Tar with Histamine | + | (1) Histamine-Tar with Histamine attractant added. |
| − | (2) Histamine-Tar with | + | (2) Histamine-Tar with Motility buffer added (control).<br> |
</p> | </p> | ||
</div> | </div> | ||
| − | + | <br><br><br> | |
| + | </div> | ||
| + | <!-- 12 text div --> | ||
| + | <div class="row"> | ||
| + | <div class="col-md-12 col-sm-12"> | ||
| + | <p class="text-justify"> | ||
| + | The results presented above, mainly proves the concept of the ability to alter the LBD of a chemoreceptor using software. | ||
| + | Moreover, these results might help with breakthroughs and lead to newly designed reporters for novel detection. | ||
| + | </p> | ||
| + | </div> | ||
</div> | </div> | ||
| Line 562: | Line 642: | ||
<!--===============--> | <!--===============--> | ||
<!-- =========== END Histamine Content =========== --> | <!-- =========== END Histamine Content =========== --> | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</div> | </div> | ||
| + | </div> | ||
| + | <!-- ======================== END: 222 ======================== --> | ||
| + | </div><!-- End: tabs --> | ||
| + | </div><!-- End: 10/12 --> | ||
| + | |||
| − | + | <!-- Referances --> | |
| − | + | <div class="row"> | |
| − | + | <div class="col-sm-8 col-sm-offset-2"><!-- 8/12 --> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| + | <p class="referances"> | ||
| + | References:<br> | ||
| + | 1. Reyes-Darias, J.A., Yang, Y., Sourjik, V., and Krell, T. (2015). Correlation between signal input and output in PctA and PctB amino acid chemoreceptor of Pseudomonas aeruginosa. Mol. Microbiol. 96, 513–525.<br> | ||
| + | <br> | ||
| + | 2. Moretti, R., Bender, B.J., Allison, B. and Meiler, J., 2016. Rosetta and the Design | ||
| + | of Ligand Binding Sites. Computational Design of Ligand Binding Proteins, pp.47-62.<br> | ||
| + | </p> | ||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
| − | |||
<br> | <br> | ||
<br> | <br> | ||
<br> | <br> | ||
| − | </div> | + | </div><!-- page wrapper --> |
| + | |||
<!--Code: Click on img to enlarge it--> | <!--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 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> | ||
| − | + | <!--Arrow up --> | |
| − | + | <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> | |
Latest revision as of 02:09, 20 October 2016
PctA-Tar chimera Introduction
One of S.Tars sub-projects focused on altering the LBD of Tar chemoreceptor in order to design new hybrid chimeras. That was accomplished by replacing the LBD of the original Tar chemoreceptor with a new one, from a different source, without modifying Tar signaling region. As a proof of concept for the newly designed Tar chimeras and the S.Tar project, we focused on testing the PctA-Tar hybrid.
Fig. 1: Scheme of native Tar chemoreceptor, native PctA receptor and PctA-Tar chimera. Adapted from (1)
PctA is a chemoreceptor found in the Pseudomonas Aeruginosa .It mediates
chemotaxis towards amino acids and away from organic compounds. It can sense all
amino acids except for Aspartate (1).
To construct this chimera, the LBD sequence of the PctA was obtained from the Pseudomonas genome database,
while the signaling region of Tar was obtained from the iGEM parts catalog
(K777000). Using these two sequences, we built a Biobrick part (K1992007)
which was tested by transforming the device to bacteria that lacks chemoreceptors -
UU1250. It is important
to note that this chimera has been constructed before in the literature (1).
Fig. 2: Biobrick device of the PctA-Tar chimera.
Test and results
As an initial step, we generated a 3D model of the PctA-Tar chimera, figure 3, using the Phyre2 Protein Fold Recognition server to assure the correct folding of both the LBD and the signaling regions.
Fig. 3: PctA-Tar chimera 3D structure. The Tar signaling regions is in gray, the PctA LBD is in red.
Following transformation, a swarming plate assay was performed in order to confirm the functionality of the hybrid receptor. A scheme of the assay is presented below (figure 4). It is important to mention that this assay was performed on BA medium as the original assay on TB medium failed. From the results seen in figure 5, it is clear that the chimera functions and controls the chemotactic ability of the bacteria leading to swarming response. This is compared to the control, UU1250 strain, that did not show any chemotactic ability as expected and no chemotactic rings were formed.
Fig. 4: a scheme for the swarming plate assay. In brief: Using a low percentage agar media plate, add a drop of bacteria to the middle of the plate. Incubate at 30 degrees for several hours. A halo or “chemotactic ring” should be formed in the agar plate.
a.
b.
c.
Fig. 5: Swarming assay for attractant response of the PctA-Tar chimera.
a. PctA chimera, b. Negative control- UU1250 strain w/o the Tar expression plasmid, c. positive control - ΔZras strain expressing all chemoreceptors.
Next, to prove that the chimera is localized in the membrane, GFP was fused to its C-terminus with a short linker sequence (K1992010), figure 6. The results seen in figure 7, show that indeed the chimera is localized to the membrane (poles).
Fig. 6: Biobrick device of the PctA-Tar chimera fused to GFP.
Fig. 7: Results of GFP fusion. (A) Positive control- E.Coli strain expressing GFP protein,(B) Negative control- UU1250 strain expressing Tar chemoreceptor, (C) UU1250 strain expressing Tar-GFP chemoreceptor, (D) UU1250 strain expressing PctA-Tar-GFP Chimera, fluorescence (490nm excitation).
Finally, in the video below is a working concept of the FlashLab project - a chip that serves as a detection tool based on the chemotaxis system of E. coli bacteria. In the video, a commercial ibidi microfluidic chip filled with a suspension of bacteria expressing the chimera and chromoprotein (J23100 + K1357009) can been seen. A solution of repellent (10-3M Tetrachloroethylene) was added to the chip and the displacement of the bacteria was monitored and recorded.
Fig. 8: A steps scheme of the FlashLab concept: Add bacteria expressing both the chemoreceptor of your choice and a chromoprotein to a fluidic chip. Add the tested sample to the chip. If the chemoreceptor detecets the substance in the sample, a displacement of the bacteria will become visible. .
In video 1, the displacement of the bacteria can be clearly seen in test chip compared to the control chip.
Video 1: from left to right: (1) PctA-Tar chimera with Tetrachloroethylene repellent added. (2) PctA-Tar chimera with Motility buffer added (control).
With the supporting evidence of the results presented above, it can be concluded that both concepts: LBD altering and the FlashLab platform, have been proven and work under real life conditions and seem promising for detection of various substances in the near future.
Histamine-Tar Introduction
The heart of the S.Tar project is the Tar chemoreceptor, one of four E. coli receptors. Our goal is to create an engineered bacteria which has chemotaxis receptors sensitive to materials other than its native ligands. We show that E. coli can be engineered to respond to completely new materials. By changing Tar’s ligand binding domain (LBD) to other LBDs from various sources or by mutating it.
Fig. 1: Sequencing Results. "Query" describes the native Tar LBD sequence and "Sbjct" describes the design mutations sequence. The mutation region is marked with color (blue or red).
The bacterial world offers a relatively small selection of chemoreceptors in comparison to
the vast number of possible ligands. These receptors evolved specifically to recognize substances
which benefit or harm the organism. On top of that the fact that the majority of known
receptors today are not well characterized meant that we had very few options of creating chimeric
receptors as we initially planned.
In light of the above we had to turn to a new path – redesigning the Tar chemoreceptor to bind a
different ligand using computational biology - The Rosetta software.
Out of the Rosetta’s 870 suggested mutations only 11 variants were eventually cloned into the native Tar ligand-binding domain (LBD).
See Computational Design page for more information regarding the design process.
Out of all the tested variants, only one was discovered to be attracted to Histamine. Sequencing results
showed that the only mutations to occur in this variant were those planned by the Rosetta’s design. The desired sequences can be seen in figure 1.
Fig. 2: Histamine-Tar filtering process scheme
Test and results
We observed the bacteria’s response to the attractant, Histamine, by using a microscope. It is evident in figure 3b that roughly 20 minutes after the addition of the Histamine, the bacteria concentration in the vicinity of the Histamine is much greater than in the the beginning of the experiment (figure 3a).
a.
b.
c.
d.
Fig. 3: Microscope results of chemotaxis activity for variant His_9 with 10mM Histamine. (a) Tar-Histamine: before adding Histamine. (b) Tar-Histamine: 20 minutes after adding Histamine. (c) Tar-Histamine: before adding the Motility buffer (control solution). (d) Tar-Histamine: 20 minutes after adding the Motility buffer.
To prove the correct localization of the Tar-Histamine, GFP was fused to its C-terminus with a short linker sequence (E0040) . The results seen in figure 4 show that indeed the chimera is localized to the membrane (poles).
Fig. 4: Results of GFP fusion. (A) Positive control- E.Coli strain expressing GFP protein, (B) Negative control- UU1250 strain expressing Tar chemoreceptor, (C) UU1250 strain expressing Tar-GFP chemoreceptor, (D) UU1250 strain expressing Histamine-Tar-GFP Chimera, fluorescence (490nm excitation).
Finally, in the video below, a working concept of the FlashLab project - a chip that serves as a detection tool based on the chemotaxis system of E. coli bacteria is presented. In the video, a commercial ibidi microfluidic chip filled with a suspension of bacteria expressing both the chemoreceptor and chromoprotein (K1992011 + K1357008) can been seen. A solution of attractant (10-3M Histamine) was added to the chip and bacteria displacement was monitored and recorded
Video 1: from left to right:
(1) Histamine-Tar with Histamine attractant added.
(2) Histamine-Tar with Motility buffer added (control).
The results presented above, mainly proves the concept of the ability to alter the LBD of a chemoreceptor using software. Moreover, these results might help with breakthroughs and lead to newly designed reporters for novel detection.
References:
1. Reyes-Darias, J.A., Yang, Y., Sourjik, V., and Krell, T. (2015). Correlation between signal input and output in PctA and PctB amino acid chemoreceptor of Pseudomonas aeruginosa. Mol. Microbiol. 96, 513–525.
2. Moretti, R., Bender, B.J., Allison, B. and Meiler, J., 2016. Rosetta and the Design
of Ligand Binding Sites. Computational Design of Ligand Binding Proteins, pp.47-62.
