Difference between revisions of "Team:Technion Israel/Design"

 
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<img src="https://static.igem.org/mediawiki/2016/d/db/T--Technion_Israel--icon_intro.png" class="img-responsive img-center cir_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 cir_tabs" width="75" height="75">
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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</b></h4>
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<br><h4 class="text-center">Design</h4>
 
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<br><h4 class="text-center"><b>Results</b></h4>
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<br><h4 class="text-center">Results</h4>
 
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<br><h4 class="text-center"><b>OutLook</b></h4>
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<br><h4 class="text-center">Conclusion</h4>
 
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<!-- =========== 1. Intro =========== -->
 
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<br>
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<h1 class="text-center"><u>FlashLab - Introduction</u></h1>
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<h2 class="">Introduction</h2>
 
<h2 class="">Introduction</h2>
 
<p class="text-justify">
 
<p class="text-justify">
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FlashLab, a novel detection tool based on the chemotaxis system of <I>E. coli</I>.  
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FlashLab is a novel detection tool based on the <a data-toggle="popover" data-trigger="click" data-original-title="Info:" data-html="true" data-content="biological system that controls the bacterial motion and induces movement away from a chemo-repellent or towards a chemo-attractant">chemotaxis system<i class="entypo-check"></i></button></a> of <I>E. coli</I> bacteria. It utilizes chemotaxis to concentrate bacteria expressing a chromo-protein, this in turn, creates a visible gradient in color – detection of a target material.  
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It uses the chemotaxis system to concentrate colored bacteria, this in turn, creates a visible gradient  
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FlashLab is an application of the S.Tar project. S.Tar is a platform for programmable chemotaxis that allows the user to select the material that will induce a bacterial chemotactic response. For more information please visit <a href="https://2016.igem.org/Team:Technion_Israel/S.Tar_intro">S.Tar page</a>.
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in color – detection of target material. Using the S.tar technology, the FlashLab can detect verity of  
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Using S.Tar technology, FlashLab can detect a variety of materials: hormones, amino acids, organic compounds etc.
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materials: hormones, amino acids, PCE etc.
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<img src="https://static.igem.org/mediawiki/2016/7/74/T--Technion_Israel--fig1.JPG" class="img-responsive img-center img-cont" style="cursor: pointer;"><br>
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<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>
 
</a>
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<p class="text-center"><b>Fig. 1:</b> Detection tool explanation</p>
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<p class="text-center"><b>Fig. 1:</b> A scheme of the FlashLab concept, add bacteria expressing the chemoreceptor of your choice and a chromo protein, to a fluidic chip. Add the sample in question to the chip. If the sample contains the substance that is recognized by the chemoreceptor, a displacement of the bacteria will become visible. If not, then no displacement will be seen.</p>
 
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<h2 class="">Design</h2>
 
<h3>FlashLab parts</h3>
 
<h3>FlashLab parts</h3>
 
<p class="text-justify">
 
<p class="text-justify">
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The device is composed of a <a href="http://ibidi.com/xtproducts/en/ibidi-Labware/sticky-Slides/sticky-Slide-I-Luer" target="_blank">commercial fluidic chip</a>:
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The device is composed of a <a href="http://ibidi.com/xtproducts/en/ibidi-Labware/sticky-Slides/sticky-Slide-I-Luer"target="_blank">commercial fluidic chip</a>:
 
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<img src="https://static.igem.org/mediawiki/2016/8/82/T--Technion_Israel--fig2.jpg" class="img-responsive img-center img-cont" style="cursor: pointer;"><br>
 
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</a>
 
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<p class="text-center"><b>Figure 2:</b>The geometry of a commercial fluidic chip.</p>
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<p class="text-center"><b>Fig. 1:</b>The geometry of a commercial fluidic chip.</p>
 
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The chip is open on the button part and closed with a standard microscope cover glass (0.3[mm] thick).
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The chip is open on the bottom part and closed with a standard microscope cover glass (0.3 [mm] thick).
 
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<h3>FlashLab setup</h3>
 
<h3>FlashLab setup</h3>
 
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<p class="text-justify">
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The setup of the device is composed of two parts, as shown below (Figure 3):
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The setup of the device is composed of two parts, as shown below (Figure 2):<br>
 +
 
 +
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<b>a. </b>The channel is filled with colored <i>E. coli</i>  bacteria suspended in motility buffer.<br>
 +
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<b>b. </b>The sample is loaded into one of the entry slots.
 +
 
 
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<img src="https://static.igem.org/mediawiki/2016/thumb/c/c1/T--Technion_Israel--fig3.jpg/800px-T--Technion_Israel--fig3.jpg" class="img-responsive img-center img-cont" style="cursor: pointer;"><br>
 
<img src="https://static.igem.org/mediawiki/2016/thumb/c/c1/T--Technion_Israel--fig3.jpg/800px-T--Technion_Israel--fig3.jpg" class="img-responsive img-center img-cont" style="cursor: pointer;"><br>
 
</a>
 
</a>
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<p class="text-center"><b>Figure 3: </b>The chip setup.</p>
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<p class="text-center"><b>Fig. 2: </b>The chip setup.</p>
 
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<div class="col-sm-12">
 
<div class="col-sm-12">
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<h3>FlashLab test</h3>
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<h3>FlashLab assay</h3>
 
<p class="text-justify">
 
<p class="text-justify">
 
Once the sample is loaded, it diffuses into the channel. If the sample contains a repellent, the bacteria  
 
Once the sample is loaded, it diffuses into the channel. If the sample contains a repellent, the bacteria  
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will react and move away from it as shown below:
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will react and flee from it as shown below:
 
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<img src="https://static.igem.org/mediawiki/2016/thumb/8/84/T--Technion_Israel--fig44.jpg/800px-T--Technion_Israel--fig44.jpg" class="img-responsive img-center img-cont" style="cursor: pointer;"><br>
 
<img src="https://static.igem.org/mediawiki/2016/thumb/8/84/T--Technion_Israel--fig44.jpg/800px-T--Technion_Israel--fig44.jpg" class="img-responsive img-center img-cont" style="cursor: pointer;"><br>
 
</a>
 
</a>
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<p class="text-center"><b>Figure 4: </b>Chemotaxis reaction in the chip.</p>
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<p class="text-center"><b>Fig. 3: </b>Chemotaxis reaction in the chip.</p>
 
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<p class="text-justify">
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This will cause changes in the bacteria concentration: very low concentration, where the repellent diffused
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The chemotactic response will result in visible changes in the bacterial concentration throughout the chip: Very low concentration in the immediate area of the slot in which the sample was loaded, adjacent to a higher concentration of bacteria created due to the fleeing bacterial population. These changes will be visible to the naked eye, as the higher concentration of colored bacteria results in darker color (blue gradient, figures 2 and 3).
−
to, next to a very high concentration, where the bacteria moved to (right picture, figure 1:4). Those changes  
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If the sample does not contain the target material, the bacteria will not react and no gradient will be formed.  
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will also be visible, as the higher concentration of colored bacteria manifests itself in a stronger color  
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<br><br>
−
(blue gradient, figures 1:3 and 1:4).<br>
+
For more information see <a href="https://2016.igem.org/Team:Technion_Israel/Model">mathematical model</a>.
−
If the sample does not contain target material, the bacteria will not react and no gradient will form.<br>
+
−
For more information see <a href="https://2016.igem.org/Team:Technion_Israel/Model" target="_blank">mathematical model</a>.
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<!-- ==================== 3: Resuls ==================== -->
 
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<br>
 
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<h1 class="text-center"><u>Results:</u></h1>
 
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<h3>FlashLab parts:</h3>
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<h2 class="">Results</h2>
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<h3>FlashLab parts</h3>
 
<p class="text-justify">
 
<p class="text-justify">
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The set up for the device is as shown in "Design and Implementation". The bacteria, <I>E.coli</I> strain  
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The setup of the device is as shown in the "Design" Tab. The bacteria, UU1250  <I>E.coli</I> strain with a cloned Tar-PctA receptor , was picked from a petri dish and suspended in motility buffer. The Chemo-repellent used was TCE (trichloroethylene) in concentration of 0.02 [M] while the control was motility buffer. The images were taken in a different times.  
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<b>#$%^&@#$%^</b>, was taken from a petri dish and diluted in 180[µL] of motility buffer. The Chemo-repellent  
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used was 30 [µL] of <b>#$%^&@#$%^</b> in concentration of α[M] while the control was motility buffer. The  
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pictures were taken in differential of  apart.
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<p class="text-center"><b>Figure XYZ:</b>ABC.</p>
 
 
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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/igem.org/c/ca/T--Technion_Israel--Model2.png" class="img-responsive img-center img-cont" width="290" style="cursor: pointer;"><br>
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</a>
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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/2016/7/76/T--Technion_Israel--ModelControl.png" class="img-responsive img-center img-cont" width="220" style="cursor: pointer;"><br>
 +
</a>
 +
</div>
 +
<div class="col-md-12 col-sm-12">
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<p class="text-center"><b>Fig. 1:</b>
 +
<b>a.</b> Chemotaxis of <i>E. coli</i> with a S.Tar PctA receptor due to exposure to TCE (enhanced picture).
 +
<b>b.</b> <i>E. coli</i> with a S.Tar PctA receptor exposed to motility buffer (control).
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<p class="text-justify">
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Following γ[min], a noticeable gradient of blue color formed in the chip's channel. on the far left, a relatively  
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After 15[min] , a noticeable color gradient is formed in the channel. In 120 [min] a relatively light shade can be seen near the entry point. Adjacent to it, an area with a darker tone and from there up to the end of the channel, the color did not change at all. This fits the theory perfectly, as the lighter shade is caused by colored bacteria moving away from the repellent. The darker shade is the clustering of bacteria in the chemo-repellent diffusion limit. All other bacteria in the channel, were not exposed to the repellent and did not react accordingly.
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light shade. Next to it, an area with a darker tone and from there up to the right end of the channel, the tone
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−
didn’t change at all. This lines up with the theory perfectly, as the lighter shade is caused by colored bacteria  
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<br>
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moving away from the repellent. The darker shade is the clustering of bacteria in the chemo-repellent diffusion  
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limit. All other bacteria in the channel, were not exposed to the repellent and so didn’t react.<br>
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<br>
 
<br>
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<b>==========For more information see chip experiment protocol==========</b>
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For more information see chip <a href="https://static.igem.org/mediawiki/2016/1/11/T--Technion_Israel--Protocol_-_Chemotaxis_on_chip_test.pdf"target="_blank">experiment protocol</a>.
 
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<!-- ==================== 4: Outlook ==================== -->
 
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<h1 class="text-center"><u>Outlook</u></h1>
 
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<h2 class="">Conclusion</h2>
 
<p class="text-justify">
 
<p class="text-justify">
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FlashLab has advatages as a detection tool:
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- <b>Cheap</b> The only major cost is the fluidic chip and one costs about 15$ and can be reuse multible times.<br>
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We developed FlashLab, the hardware of the S.Tar platform. S.Tar has the potential to offer a wide library of “expert” strains for the detection of materials that do not naturally induce chemotactic movement in the native <i>E. coli</i>. This was obtained using synthetic biology tools, including computational design which allowed us to create new mutations in the Tar chemoreceptor.  
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- <b>Fast</b>  as shown in the expermints, it takes about 30 minutes for detection. This is faster then most other bacterial detection (based on transecription and translation) and most laboratory tests (HPLC). <br>
+
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- <b>Verstile</b> The S.tar system enable this device to detect verity of materials: hormones, amino acids, PCE etc. <br>
+
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- <b>Senstive:</b> Bacteria can sense extrimly small traces of target material.<br>
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- <b>Easy to use:</b> The set up of the system is an easy, two parts process. To reuse the chip you only need to flush the channel with water and dry.<br>
+
 
<br>
 
<br>
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<b>======(For Farther improvement see "Design and Devolpment")======</b>
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<br>
 +
FlashLab is a tool which combines both biological and mechanical aspects. As we mentioned before, computational tools were required as well in order to develop the S.Tar platform. This multidisciplinary innovation was achieved thanks to the different backgrounds of our team members. Our team is comprised of students from the faculty of Biotechnology that oversaw the Tar chemoreceptor modification in the wet lab, students from the faculties of Mechanical engineering and Chemical engineering that developed the mathematical model for the flow and chemotaxis profiles inside the microchannel and students from the faculties of Electrical engineering and Computer Science that were in charge of the computational design aspects of the project. The unique composition of our team was crucial to the construction of a complex but elegant system.
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<h2 class="">Advantages</h2>
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<p class="text-justify">
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<br>
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There are several methods for detection of small molecules. The following table summarizes the most common methods in the field:
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    <table class="table table-list-search">
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                    <thead>
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                        <tr>
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                            <th>Method</th>
 +
                            <th>Description</th>
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                            <th>Disadvantages</th>
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                        </tr>
 +
                    </thead>
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 +
                    <tbody>
 +
 +
    <tr>
 +
                            <td>Reporter genes <b>(2)</b></td>
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                            <td>Fusion of a reporter gene to a biological biobrick which is regulated by a material of interest. </td>
 +
                            <td>Requires gene expression and translation.<br>The material of interest must penetrate the bacterial membrane. </td>
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                        </tr>
 +
 
 +
                        <tr>
 +
                            <td>Chromogenic assay <b>(3)</b></td>
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                            <td>A chemical reaction which results in the change of absorbance of a certain molecule when it interacts with the analyte.</td>
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                            <td>Specific for very limited range of molecules.<br>Expensive.</td>
 +
                        </tr>
 +
 
 +
                        <tr>
 +
                            <td>ELISA- Immunofluorescence assay <b>(4)</b></td>
 +
                            <td>A specific interaction between an antibody and its antigen is coupled to an enzymatic fluorescent reaction.</td>
 +
                            <td>Specific antibodies are required.<br>Expensive.<br>Requires dedicated equipment.</td>
 +
                        </tr>
 +
 +
                        <tr>
 +
                            <td>HPLC <b>(5)</b></td>
 +
                            <td>Chromatographic separation based on polarity.</td>
 +
                            <td>Requires dedicated equipment and expertise.<br>Expensive</td>
 +
 +
                    </tbody>
 +
                </table> 
 +
 
 +
<br>
 +
<br>
 +
<br>
 +
<p class="text-justify">
 +
FlashLab advantages as a detection tool:
 +
<br>- <b>Cost effective</b>: The only major cost is the fluidic chip which costs about 15$ and can be reused multiple times.
 +
<br>- <b>Fast</b>: as shown in the experiments, the detection takes about 30 minutes. This is faster than most other bacterial based detection that depends on transcription and translation.
 +
<br>- <b>Versatile</b>: The S.Tar system enables this device to detect a variety of materials: hormones, amino acids, organic compounds etc.
 +
<br>- <b>User friendly</b>: The setup of the system is an easy two part process.<br>
 +
FlashLab, offers a fast, cheap, easy to use and versatile detection.<br>
 +
<br>
 +
<br>
 +
 
 
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References:<br>
 
References:<br>
−
1. KELLER, Evelyn F.; SEGEL, Lee A. Model for chemotaxis. Journal of theoretical biology, 1971, 30.2: 225-234.‏‬ <br>
+
1. MAZZAG, B. C.; ZHULIN, I. B.; MOGILNER, Alexander. Model of bacterial band formation in aerotaxis. Biophysical journal, 2003, 85.6: 3558-3574.<br><br>
 +
2. Naylor, Louise H. "Reporter gene technology: the future looks bright."Biochemical pharmacology 58.5 (1999): 749-757.<br>
 +
3. Santos-Figueroa, Luis E., et al. "Chromogenic and fluorogenic chemosensors and reagents for anions. A comprehensive review of the years 2010–2011." Chemical Society Reviews 42.8 (2013): 3489-3613.‏<br><br>
 +
4. Blake, Christopher, and Barry J. Gould. "Use of enzymes in immunoassay techniques. A review." Analyst 109.5 (1984): 533-547.<br><br>‏
 +
5. Kucharska, Marta, and Jan Grabka. "A review of chromatographic methods for determination of synthetic food dyes." Talanta 80.3 (2010): 1045-1051.‏<br><br>‏
 
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Latest revision as of 23:24, 19 October 2016

S.tar, by iGEM Technion 2016

S.tar, by iGEM Technion 2016

Introduction

FlashLab is a novel detection tool based on the chemotaxis system of E. coli bacteria. It utilizes chemotaxis to concentrate bacteria expressing a chromo-protein, this in turn, creates a visible gradient in color – detection of a target material. FlashLab is an application of the S.Tar project. S.Tar is a platform for programmable chemotaxis that allows the user to select the material that will induce a bacterial chemotactic response. For more information please visit S.Tar page. Using S.Tar technology, FlashLab can detect a variety of materials: hormones, amino acids, organic compounds etc.


Fig. 1: A scheme of the FlashLab concept, add bacteria expressing the chemoreceptor of your choice and a chromo protein, to a fluidic chip. Add the sample in question to the chip. If the sample contains the substance that is recognized by the chemoreceptor, a displacement of the bacteria will become visible. If not, then no displacement will be seen.


Design

FlashLab parts

The device is composed of a commercial fluidic chip:


Fig. 1:The geometry of a commercial fluidic chip.

The chip is open on the bottom part and closed with a standard microscope cover glass (0.3 [mm] thick).



FlashLab setup

The setup of the device is composed of two parts, as shown below (Figure 2):
      a. The channel is filled with colored E. coli bacteria suspended in motility buffer.
      b. The sample is loaded into one of the entry slots.


Fig. 2: The chip setup.

FlashLab assay

Once the sample is loaded, it diffuses into the channel. If the sample contains a repellent, the bacteria will react and flee from it as shown below:


Fig. 3: Chemotaxis reaction in the chip.

The chemotactic response will result in visible changes in the bacterial concentration throughout the chip: Very low concentration in the immediate area of the slot in which the sample was loaded, adjacent to a higher concentration of bacteria created due to the fleeing bacterial population. These changes will be visible to the naked eye, as the higher concentration of colored bacteria results in darker color (blue gradient, figures 2 and 3). If the sample does not contain the target material, the bacteria will not react and no gradient will be formed.

For more information see mathematical model.

Results

FlashLab parts

The setup of the device is as shown in the "Design" Tab. The bacteria, UU1250 E.coli strain with a cloned Tar-PctA receptor , was picked from a petri dish and suspended in motility buffer. The Chemo-repellent used was TCE (trichloroethylene) in concentration of 0.02 [M] while the control was motility buffer. The images were taken in a different times.

a.

b.

Fig. 1: a. Chemotaxis of E. coli with a S.Tar PctA receptor due to exposure to TCE (enhanced picture). b. E. coli with a S.Tar PctA receptor exposed to motility buffer (control).

After 15[min] , a noticeable color gradient is formed in the channel. In 120 [min] a relatively light shade can be seen near the entry point. Adjacent to it, an area with a darker tone and from there up to the end of the channel, the color did not change at all. This fits the theory perfectly, as the lighter shade is caused by colored bacteria moving away from the repellent. The darker shade is the clustering of bacteria in the chemo-repellent diffusion limit. All other bacteria in the channel, were not exposed to the repellent and did not react accordingly.

For more information see chip experiment protocol.

Conclusion

We developed FlashLab, the hardware of the S.Tar platform. S.Tar has the potential to offer a wide library of “expert” strains for the detection of materials that do not naturally induce chemotactic movement in the native E. coli. This was obtained using synthetic biology tools, including computational design which allowed us to create new mutations in the Tar chemoreceptor.

FlashLab is a tool which combines both biological and mechanical aspects. As we mentioned before, computational tools were required as well in order to develop the S.Tar platform. This multidisciplinary innovation was achieved thanks to the different backgrounds of our team members. Our team is comprised of students from the faculty of Biotechnology that oversaw the Tar chemoreceptor modification in the wet lab, students from the faculties of Mechanical engineering and Chemical engineering that developed the mathematical model for the flow and chemotaxis profiles inside the microchannel and students from the faculties of Electrical engineering and Computer Science that were in charge of the computational design aspects of the project. The unique composition of our team was crucial to the construction of a complex but elegant system.




Advantages


There are several methods for detection of small molecules. The following table summarizes the most common methods in the field:


Method Description Disadvantages
Reporter genes (2) Fusion of a reporter gene to a biological biobrick which is regulated by a material of interest. Requires gene expression and translation.
The material of interest must penetrate the bacterial membrane.
Chromogenic assay (3) A chemical reaction which results in the change of absorbance of a certain molecule when it interacts with the analyte. Specific for very limited range of molecules.
Expensive.
ELISA- Immunofluorescence assay (4) A specific interaction between an antibody and its antigen is coupled to an enzymatic fluorescent reaction. Specific antibodies are required.
Expensive.
Requires dedicated equipment.
HPLC (5) Chromatographic separation based on polarity. Requires dedicated equipment and expertise.
Expensive



FlashLab advantages as a detection tool:
- Cost effective: The only major cost is the fluidic chip which costs about 15$ and can be reused multiple times.
- Fast: as shown in the experiments, the detection takes about 30 minutes. This is faster than most other bacterial based detection that depends on transcription and translation.
- Versatile: The S.Tar system enables this device to detect a variety of materials: hormones, amino acids, organic compounds etc.
- User friendly: The setup of the system is an easy two part process.
FlashLab, offers a fast, cheap, easy to use and versatile detection.




References:
1. MAZZAG, B. C.; ZHULIN, I. B.; MOGILNER, Alexander. Model of bacterial band formation in aerotaxis. Biophysical journal, 2003, 85.6: 3558-3574.

2. Naylor, Louise H. "Reporter gene technology: the future looks bright."Biochemical pharmacology 58.5 (1999): 749-757.
3. Santos-Figueroa, Luis E., et al. "Chromogenic and fluorogenic chemosensors and reagents for anions. A comprehensive review of the years 2010–2011." Chemical Society Reviews 42.8 (2013): 3489-3613.‏

4. Blake, Christopher, and Barry J. Gould. "Use of enzymes in immunoassay techniques. A review." Analyst 109.5 (1984): 533-547.

‏ 5. Kucharska, Marta, and Jan Grabka. "A review of chromatographic methods for determination of synthetic food dyes." Talanta 80.3 (2010): 1045-1051.‏

‏



S.tar, by iGEM Technion 2016