Team:EPF Lausanne

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<h1 class="cntr"> Our project in a nutshell</h1>
<h1 class="cntr"> Our project in a nutshell</h1>
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<figcaption class="cntr">Association of split IFP 1.4 fragments</figcaption></div>
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The 2014 EPFL iGEM team has been working on showing that biologically engineered contexts can detect and process signals in fast and efficient ways. With this in mind, our team worked on  bringing forward a novel idea: combining Protein Complementation techniques to Biosensors to achieve fast spatiotemporal analysis of bacterial response to stimuli.
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The 2014 EPFL iGEM team has been working on showing that biologically engineered organisms can detect and process signals quickly and efficiently. With this in mind, our team brought forward a novel idea: combining protein complementation techniques with biosensors to achieve fast spatiotemporal analysis of cell responses to stimuli. In other words, we fused complementary reporter protein fragments to interacting proteins. The presence of a given stimulus leads to the interaction of the proteins of interest thus allowing the fused split complements to re-acquire their functional conformation and emit signal. We thereby are able to detect signal dynamics by relying on much faster post-transcriptional modifications rather than slow traditional reporter transcription.
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we are able to detect signal dynamics thanks to the association of the reporter fragments. We thereby rely on much faster post-transcriptional modifications to generate signals rather than traditional reporter transcription.
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The principle is the following: two complementary fragments of a reporter protein are fused to interacting proteins. When the interaction is stimulated, the two fragments associate, thereby reconstituting the reporter signal in a much faster way than traditional post-transcriptional reporters.
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As a proof of concept of this idea, we aimed to engineer the Cpx Pathway – an E.Coli endogenous two component regulatory system responsive to periplasmic stress to develop a BioPad: a biological TouchPad made of touch responsive bacteria in a microfluidic chip allowing the control of electronic devices . </p>
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As a proof-of-concept, we aimed to develop the first BioPad: a biological trackpad made of a microfluidic chip, touch-responsive organisms and a signal detector. To make our organisms touch-sensitive, we engineering two stress-related pathways in <i>E. coli</i> and <i>S. cerevisiae</i>.<!--In <i>E. coli</i>, we engineered the Cpx Pathway - a two-component regulatory system responsive to envelope stress. In <i>S. cerevisiae</i>, we modified the HOG Pathway - a MAPKK pathway responsive to osmotic stress.--> As for the reporter proteins, we worked mainly with fluorescent proteins but also implemented a split luciferase complementation assay. To learn more about the various components of our project, check out our <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Overview">overview section</a>, as well as the different <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Parts">parts</a> submitted by our team. If you are a judge, you might also be interested in our <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Results">results page</a>, our <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Data">data page</a> and our <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Judging">judging form</a>. </p>
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<h2>Why a BioPad?</h2>
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<p class="lead"> The biological concepts behind the BioPad project have applications both in basic and applied sciences. From a purely scientific perspective, the ideas introduced and implemented by our project are novel and promising for future applications. The BioPad is also an attractive concept that is tangible for the general public and will allow people to look at synthetic biology in a different way. Hence, the combination of novel biological concepts, a cool idea, and the community awareness that our project provides, makes the BioPad project perfect for iGEM !</p>
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<p class="lead">The biological concepts behind the BioPad project have applications in basic and applied sciences. From a scientific perspective, the ideas introduced and implemented by our project are novel and promising for future applications. The BioPad is also an interesting concept that will encourage public awareness of synthetic biology. The tangibility of the project will allow the general public to look at synthetic biology in a better way, as people will understand how great genetically modified organisms are! To get down the basics, the combination of novel biological concepts, a cool idea, and the community awareness that our project provides, makes the BioPad project perfect an ideal project for iGEM!
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  <p class="lead">With respect to basic sciences, our system serves as a good proof that protein complementation techniques are suitable for applications in the context of biosensors – especially for two component regulatory systems. The introduction of the split IFP1.4 into the registry will allow future iGEM and research teams to take advantages of the reversibility and precision of this protein. Moreover, our work on the Cpx pathway will allow future iGEM teams to make us of other members of the OmpR/PhoB subfamily as well as other two-component regulatory systems in new ways.
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  <p class="lead">With respect to basic sciences, the BioPad demonstrates that protein complementation techniques are suitable for biosensors – especially for two-component regulatory systems. The introduction of the split IFP1.4 (infrared fluorescent protein) into the registry will allow future iGEM and research teams to take advantage of the reversibility and precision of this protein. Moreover, our work on the Cpx pathway will allow future iGEM teams to make novel uses of other members of this subfamily, as well as other two-component regulatory systems.  
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As for applied sciences, the potential uses of the BioPad include the delivery of a cheap, fast, efficient, and accurate antibiotic screening systems enabling an easy way to quantify how antibiotics affect the periplasm in gram negative bacteria; the BioPad project could also be the source of an "antibiotic complement" drug allowing
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As for applied sciences, the BioPad could be used to deliver a cheap, fast, efficient, and accurate antibiotic screening system allowing researchers to easily quantify the effects of antibiotics on gram-negative bacteria. The BioPad project could also be the source of an "antibiotic complement" drug increasing the efficiency of pre-existing antibiotics. Moreover, the Biopad could provide a new approach to studying genes by allowing researchers to examine the relationship between genes and their corresponding activating signals. To learn more about the applications of our project click <a target="_blank" href="https://2014.igem.org/Team:EPF_Lausanne/Applications">here</a>.</p>
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could also provide a new way to study genes by allowing the examine the relationship between genes and their corresponding activating signals</p>
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The process by which a signal is detected upon touch starts of from our self-designed PDMS microfluidic chip: the BioPad. The BioPad is made of hundreds of compartments that represent the "pixels" of our pad. Each chamber has dimensions of 30µm x 30µm x 3µm allowing the BioPad to have single layers of E.Coli. When the surface of the chip is touched, a deformation of the chip - and thus of the chambers - leads to cellular membrane shear stress and protein aggregation/misfolding in the periplasm. The aggregated/misfolded proteins are then sensed by the sensor histidine kinase CpxA that auto-phosphorylates and transfers its phosphate to its corresponding relay protein CpxR. Upon phosphorylation, CpxR homo-dimerizes. Our engineered bacteria contain CpxR proteins fused to split reversible fluorescent or luminescent protein fragments (IFP1.4 or firefly luciferase) via a 10 amino acid 2 x GGGS flexible linker. Therefore our engineered bacteria allow us to detect CpxR dimerization, synonymous periplasmic stress and touch. Then, a self built detector made of a raspberry pi, an inexpensive CMOS, and a couple of lenses, identifies and processes the position of the light/fluorescence emitted by the BioPad. This information about the position of the light relative to chip is then used to control the associated electronic device.
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Our biological Touch Pad will allow to control electronic devices by emitting light at the specific location where the Pad has been touched. Light emission is possible by engineering reporter proteins such as the firefly Luciferase, the Renilla luciferase, the Infrared fluorescent proteins, and even the superfolder GFP.
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We split the reporter proteins and fused them to an E.Coli endogenous protein involved in the regulation of extracytoplasmic stress. The protein of interest is CpxR, a component of the CpxA-CpxR two-component regulatory system.<br /><br /> -->
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On top of developing the biological components allowing fast response to stimuli, we engineered a small, cheap and easy to use “microscope” mainly made of a small camera to detect the position of the emitted light, process the information and instruct the associated electronic device that the user is touching the BioPad a given position.
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The natural function of the CpxA­-CpxR two component regulatory system in bacteria is to control the expression of ‘survival’ genes whose products act in the periplasm to maintain membrane integrity. This ensures continued bacterial growth even in environments with harmful extracytoplasmic stresses.
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The CpxA-­CpxR two component regulatory system belongs to the class I histidine kinases and includes three main proteins:
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  <dd>an integral inner­-membrane sensor kinase, which activates and auto­phosphorylates when sensing misfolded proteins in the E.Coli periplasm. CpxA transduces its signal through the membrane to activate the cytoplasmic CpxR response regulator by a phosphotransfer reaction.</dd>
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  <dd>CpxA’s corresponding cytoplasmic response regulator belongs to the OmpR/PhoB family of winged­helix­turn­helix transcriptional response regulators and is phosphorylated by CpxA in the presence of extracytoplasmic stresses. Phosphorylation induces CpxR’s homo­dimerization, and activation as a transcription factor. Phosphorylated CpxR then binds to the promoters of genes coding for several protein folding and degradation factors that operate in the periplasm.</dd>
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  <dd>an inhibitor of CpxA that we suspect to actively compete with misfolded proteins (CpxP is a chaperone).</dd>
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  <h2 class="section-heading">Engineering: CpxR and split complementation techniques</h2>
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<!-- ENGINEERING CPXR -->
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The main component that we wish to engineer is CpxR. It has been reported that the protein homo-dimerizes upon activation. We thus plan to use fused split proteins of fluorescent and bioluminescent nature to detect its activation. <br /><br />
+
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+
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As a preliminary step, we used two fluorescent proteins sfGFP and IFP to characterisation CpxR. Split sfGFP (superfolder GFP) is an irreversible split system which will be used to prove the dimerization of CpxR. Split IFP on the other hand (Infrared Fluorescent Protein) is a reversible split system which will be used to understand the spatio-temporal dimerization of CpxR and thus allow us to better understand the On/Off mechanism of this system.<br /><br />
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To achieve our final goal, we will engineer split bioluminescent proteins: Firefly and Renilla split Luciferases. These constructs will be the main component of our system. When fused to CpxR, we are expecting to witness emission upon touch.
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  <h2 class="section-heading">Engineering: Microfluidic chip interface</h2>
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Our specially designed microfluidic chip, hereafter known as BioPad Chip, will allow easy and accurate induction of fluorescent or bioluminescent signals. The chip is made up of thousands of compartments – representing the pixels of our device ­- of the height of a bacteria. The BioTouch Chip thus allows the effective trapping and induction of stress onto our engineered bacteria. <br /><br />
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<h2 class="section-heading">Engineering: The BioPad Detector</h2>
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The signals induced by the BioTouch Chip are then processed by our self designed detection system: the BioTouch Detector. The BioTouch Detector is mainly made of a cheap computer (Raspberry Pi), a highly sensitive digital camera with appropriate light filters, and a light emitting source. The BioTouch Detector locates signals from various sources (infrared fluorescence, green fluorescence and luminescence), processes them and sends back the relative positions of the signals with respect to the BioTouch Pad. Thanks to this position, we are able to extract information such as giving a computer operating system that the position represents the position of the mouse on a screen, that the well at the given position is a suitable antibiotic candidate, or that a gene of interest has been activated. We therefore effectively control a computer or any other electronic device through a living interface: the BioTouch Pad.
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    <p>The biopad is not the only application of our modified organisms and microfluidic devices. </p>
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    <h1>Antibiotic screening device</h1>
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    <p>Bacterial envelopes are often remodeled when encountering hosts. These changes lead to the synthesis of complex envelope structures that are important virulence factors. Improper assembly of these structures can harm the bacterial envelope and lead to Extracytosolic Stress. Bacteria counter the potential envelope stresses by downregulating these virulence factors.</p>
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<p>Taking into consideration the close involvement of virulence factors and bacterial survival, the CpxA-R pathway has been shown to be a promising candidate as an antibiotic. When activated, the CpxA-R pathway activates a bacterial survival response which among other things, regulates and monitors the biogenesis of complex surface virulence factors such as pili/fimbiae and type III and type IV secretion systems. Equivalently, it has also been suggested that the CpxA-R system is involved in antibiotic mediated bacterial cell death. Our device would therefore allow us to detect and mesure activation of the CpxA-R system in real-time and thus assess the strength and influence of antiobiotics and antiobiotic candidates on the CpxA-R system. </p>
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<p>The ultimate goal of this application would thus be to allow high-throughput screenings for antibiotic candidates enabling the removal of virulence factors from pathogenic bacteria. This would improve antibiotic treatment and serve as an “antibiotic complement”.</p>
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<h1></h1>
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<p>Another application to our BioPad organisms would be related to cancer. In modern research, tumor progression is fairly difficult to evaluate: most scientists rely on the size of a tumor to understand how developed it is. Our idea would be to integrate our engineered organisms within the tumor's cellular matrix (Matrigel) to allow researchers to be able to assess the progression of tumors by how luminescent the tumor is when a light emitting molecule -luciferin- is injected. This would allow scientists to reduce unnecessary animal sacrifices in tumor research.</p>
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      <div class="back">
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  <p class="lead">Find out how we took advantage of the Cpx pathway and split IFP1.4 to give birth to bacteria emitting fast signals in response to chemical and mechanical stresses!</p>
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  <p class="lead">Discover how we engineered the HOG osmotic response pathway to create touch sensitive yeast strains! Learn more on how we implemented a split GFP and a split Luciferase in <i>S. cerevisiae</i> leading to light emission when pressure is applied.</p>
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        <h1>Microfluidics</h1>
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 +
        <!-- back content -->
 +
  <p class="lead">Our Biopad is implemented in a microfluidic chip. This tool allows all kinds of analytical experiments
 +
  and is increasingly used in biological research. From fabrication to applications, find out more about
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<div id="parts">
+
   this awesome device here!</p>
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<h1 class="cntr">PARTS</h1>
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<h2 class="section-heading">Parts submitted by the 2014 EPFL iGEM team</h2>
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<p class="lead">
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Our team submitted a total of XXX Biobricks.
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    <th class="tg-031e">Biobrick</th>
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    <th class="tg-031e">What it is ?</th>
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    <th class="tg-031e">What it does</th>
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    <th class="tg-031e">Why use it ?</th>
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    <td class="tg-031e">BBa_K1486000</td>
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<h1 class="cntr"> RESULTS </h1>
+
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<br /><br />
+
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<br /><br />
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+
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<h1> <b>Experiment 1: </b> Promoter characterisation and folding ability of fused GFP to CpxR via 10 amino acid 2 x (GGGGS) flexible linker </h1>
+
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+
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<p>Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cursus faucibus, tortor neque egestas augue, eu vulputate magna eros eu erat. Aliquam erat volutpat. Nam dui mi, tincidunt quis, accumsan porttitor, facilisis luctus, metus</p>
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<br /><br />
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<h1><b>Experiment 2: </b>CheY/CheZ fused to split firefly/renilla luciferase, and full firefly/renilla luciferase characterisation </h1>
+
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<p>Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cursus faucibus, tortor neque egestas augue, eu vulputate magna eros eu erat. Aliquam erat volutpat. Nam dui mi, tincidunt quis, accumsan porttitor, facilisis luctus, metus</p>
+
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<br /><br />
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<h1><b>Experiment 3: </b>CpxR dimerization & Dimerization Orientation </h1>
+
-
 
+
-
<p>
+
-
CpxR is the relay protein in the CpxAR two component regulatory system. It has been shown by split beta galactosidase assay that CpxR dimerizes when phosphorylated (activated) in yersinia pseudotuberculosis. Moreover, following other in vitro FRET studies, it was shown that CpxR interacted with itself.  We therefore hypothesised that dimerization would also be true in vivo in E.Coli.
+
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+
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<br /><br />
+
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+
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To determine this, we built four constructs with the various possible orientations that the split IFP1.4 fragments could have with CpxR. As shown in the graph bellow, we successfully proved that CpxR dimerized in vivo and that dimerization led to close interaction of its C-terminus.
+
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+
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<br /><br />
+
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+
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This finding is important as CpxR is part of the highly conserved OmpR/PhoB subfamily - especially for their C-terminus. This system could be used to study various other components of the OmpR/PhoB subfamily and thus lead to a new generation of highly senstitive and reactive biosensors.
+
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<div class="container cntr">
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<img src="https://static.igem.org/mediawiki/2014/c/c2/KCL_Construct_Comparison.jpg" alt="Construct Comparison">
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</div>
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+
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<p>
+
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As seen in the graph, induction of the signal was done at minute 24 (marked via a vertically spoted line). It is to be noted that the signal is immediate (3 fold increase in 2 minutes) and that the signal overall increased 30-fold.
+
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</p>
+
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+
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<br /><br />
+
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+
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<h1><b> Experiment 4: </b>Signal induction by various concentrations of KCl & signal shutdown by centrifugation </h1>
+
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+
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<p>
+
-
Having found that KCl was a good signal inducer for our signal, we decided to characterise our biobrick by testing if the signal could be modulated by various concentrations of KCl and if we were able to remove the signal by centrifugation and medium change. To do so, we read  our signal for 20 minutes without stress and then added KCl. At minute 144 we then centrifuged our cells and replaced the medium with PBS.
+
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<img src="https://static.igem.org/mediawiki/2014/6/61/KCL_titration_green_small_EPFL.jpg" alt="GA1 Shutdown">
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</div>
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+
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<p>
+
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As seen in the figure above, we successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to shut the signal down, thus proving the reversibility of our system.
+
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<!-- THE TEAM -->
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<div class="span6"><h1 class="cntr">MEET OUR TEAM</h1>
+
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<p>We are a group of 14 students from the faculties of Life, Biomechanical, and Computer Sciences, and are supervised by 2 EPFL professors, 1 Lecturer and 5 PhD students.</p></div>
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                <h4>Ted Baldwin</h4>
+
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                <h5>“I’m pretty confident that it should work.”</h5>
+
-
                <p>&nbsp;</p>
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                <h4>Içvara Barbier</h4>
 
-
                <h5>“010000110110111 101101110011011 100110000101110 01001100100”</h5>
 
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                <h4>Romane Breysse</h4>
 
-
                <h5>“I'll be 5/8 of an hour late”</h5>
 
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                <h4>Jin Chang</h4>
 
-
                <h5>Bachelor Life Sciences</h5>
 
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            <h4>Axel de Tonnac</h4>
 
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            <h5>“I forgot to eat today”</h5>
 
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            <h5>"I can't do sh*t with I'm sorry"</h5>
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-
            <h5>* Insert one of his random and very strange quotes *</h5>
 
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            <h4>Nikolaus Huwiler</h4>
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            <h5>“Imagine a world where technology is alive”</h5>
+
        <!-- front content -->
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        <h1>Hardware</h1>
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  <p class="lead">In order to process our data quickly and automatically, we built an interface with a Raspberry Pi and a camera. Discover how <br />it works by clicking here!</p>
-
            <h4>Sakura Nussbaum</h4>
+
      </div>
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            <h5>"Hello"</h5>
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            <h4>Lucie Petetin</h4>
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            <h5>"Ooh, it's sooo cuuute"</h5>
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            <h4>Cécile Piot</h4>
+
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            <h5>“The PCR didn't work... again”</h5>
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            <p>&nbsp;</p>
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            <h4>Ione Pla</h4>
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            <h5>“How about no”</h5>
+
        <!-- front content -->
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            <p>&nbsp;</p>
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      <img src="https://static.igem.org/mediawiki/2014/1/15/Human_pract_blanc.png" alt="Human practice" />
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        <h1>Human practice</h1>
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    <a href="#"><img src="https://static.igem.org/mediawiki/2014/c/ce/Img-11.png" alt=""></a>
+
  <p class="lead">Our project is well suited to show the general public the power of synthetic biology. Find out how we introduced this domain to the younger generation, and how they developed their own mini iGEM projects to tackle everyday problems with enthusiasm and creativity.</p>
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            <h4>Grégoire Repond</h4>
+
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            <h5>Parafilm specialist</h5>
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            <h4>Thomas Simonet</h4>
+
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            <h5> ''Oh no, there are no chocolate muffins left''</h5>
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        <!-- front content -->
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              <h4>Maroun Bousleiman</h4>
 
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              <h5>"This is my limit; look, I'm not smart, I'm not funny"</h5>
 
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              <p>&nbsp;</p>
 
       </div>
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 +
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 +
        <!-- back content -->
 +
<p class="lead">The first microfluidic design that provides<br /> total on-chip waste decontamination: discover<br /> how we tackled biosafety issues by<br /> engineering an awesome device!</p>
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              <h4>Oleg Mikhajlov</h4>
 
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              <h5>"Is everything alright, guys?"</h5>
 
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              <h4>Ekatarina Petrova</h4>
 
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              <h5>Thingy = coalenterazine</h5>
 
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              <p>&nbsp;</p>
 
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              <h5>"We [the TAs] also have a life!"</h5>
 
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              <h5>"Make a list"</h5>
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              <h5> “Do you talk about iGem when you meet the guys?”</h5>
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              <h4>Prof. Sebastian Maerkel</h4>
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              <h5>“I'm thinking about science”</h5>
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<div class="span6"><h1 class="cntr">MEET OUR TEAM</h1>
 +
<p>We are a group of 13 students from the faculties of Life Sciences & Technologies and Computer Sciences, </br>and are supervised by 2 EPFL professors, 1 Lecturer and 5 PhD students.</p></div>
 +
 
 +
<a href="https://2014.igem.org/Team:EPF_Lausanne/Team"><img src="https://static.igem.org/mediawiki/2014/2/2c/Team_pic_sitting.jpg" alt="the team's students" class="img-left img-border"></a>
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Latest revision as of 03:50, 18 October 2014

Our project in a nutshell




EPFL_interaction_IFP_cartoon
Association of split IFP 1.4 fragments

The 2014 EPFL iGEM team has been working on showing that biologically engineered organisms can detect and process signals quickly and efficiently. With this in mind, our team brought forward a novel idea: combining protein complementation techniques with biosensors to achieve fast spatiotemporal analysis of cell responses to stimuli. In other words, we fused complementary reporter protein fragments to interacting proteins. The presence of a given stimulus leads to the interaction of the proteins of interest thus allowing the fused split complements to re-acquire their functional conformation and emit signal. We thereby are able to detect signal dynamics by relying on much faster post-transcriptional modifications rather than slow traditional reporter transcription.

As a proof-of-concept, we aimed to develop the first BioPad: a biological trackpad made of a microfluidic chip, touch-responsive organisms and a signal detector. To make our organisms touch-sensitive, we engineering two stress-related pathways in E. coli and S. cerevisiae. As for the reporter proteins, we worked mainly with fluorescent proteins but also implemented a split luciferase complementation assay. To learn more about the various components of our project, check out our overview section, as well as the different parts submitted by our team. If you are a judge, you might also be interested in our results page, our data page and our judging form.

MEET OUR TEAM

We are a group of 13 students from the faculties of Life Sciences & Technologies and Computer Sciences,
and are supervised by 2 EPFL professors, 1 Lecturer and 5 PhD students.

the team's students

Sponsors