Team:UPO-Sevilla/Project

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<h1>Abstract</h1>
<h1>Abstract</h1>
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<p>As known, bacteria are able to respond to multiple stimuli through a collection of chemoreceptors which can sense different sensory modalities and produce chemotactic responses as a cooperative effort. Chemotaxis process is regulated by a high conserved transduction-signal system, which can be modeled and analyzed as a interconnected network of protein interactions. This system represent a great example of how cellular circuits evolve to generate functional outputs. In chemotaxis, whereas signal-transduction system is completely regulated at genetically level, flagella regulation depends on genetic expression and protein function.</p>
 
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<p>In this second circuit, our main aim is concentrate a significant population of bacteria around some vegetable polysaccharidic surface. This phenomenon was renamed as BACTERIAL CROWDING. Therefor, it is necessary that the process of interaction with the surface by a few number of bacteria triggered the production and excretion of chemicals that, acting as chemoattractants, generate a chemical-diffusing gradient which could induce chemotactic process of close bacteria. These bacteria will redirect their random movements to the same plant surface and the concentration of the cell population will raise up in this region, thanks to amplification process.</p>
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<p>The aim of our project is to concentrate a significant population of bacteria around a vegetable polysaccharidic surface, a <strong>non-difussible signal</strong>. This phenomenon was renamed as Bacterial Crowding. Therefore, it is necessary that the process of interaction with the surface by a few number of bacteria triggered the production and excretion of chemicals that, acting as <strong>chemoattractants</strong>, generate a chemical-diffusing gradient which could induce chemotactic process of close bacteria. These bacteria will redirect their random movements to the same plant surface and the concentration of the cell population will raise up in this region, thanks to an amplification process.</p>
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<p>   To that effect, it can be used two transduction signal circuits, in which PrhI-dependent promoter PprhJ will activate specifically chemoattractant production (aspartate, glutamate or salicylate), in the same way that FecI-dependent promoter PfecA. And we compare the two circuits with Fec circuit, which is common in <i>E. coli</i>. The four possible circuits are represented below. However, we will only use one: a fusion protein made up from PrhA-FecA is expected to interact with the membrane protein FecR, and triggered chemoattractant production due to vegetable polysaccharidic contact.  
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<p>In order to get that effect it is necessary to use the <strong>Prh system</strong>, the only known sensing system able to detect non-diffusble signals. We also used the Fec system to design four signal transduction circuits which would finish with PrhI-dependent promoter PprhJ, which would activate specifically chemoattractant production (aspartate, glutamate or salicylate); or with FecI-dependent promoter PfecA, which would act in the same way. You can see these circuits <a href="http://2010.igem.org/Team:UPO-Sevilla/Biobricks/Circuits" target="_blank">here</a>. However, we finally only focused on one signal transduction circuit which uses PrhA/FecA outer membrane fusion protein, performing an <strong>hybrid sensing system</strong>.</p>
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<p>We will also make use of Tar, an innate <i>E. coli</i> chemotactic receptor, which is divided in two transmembrane sequences, constituting a periplasmic domain and a cytoplasmic domain. Periplasmic domain has a dimeric structure with four helix, that binds to aspartate and glutamate asymmetrically. Conformational changes generated by such union are transmitted to cytoplasmic region, highly conserved, responsible of the activation of the flagellar motor. If we use salicylate as chemoatracttant, the system will be composed by two bacteria: <i>E. coli</i> (detection population) and <i>P. putida G7</i> (chemotactic population), because the first one is not chemotactic to salicylate.
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<p>This project could use one or two bacterial strains, depending on the chemoattractant. If it is aspartate or glutamate it would be necessary only an <i>E. coli</i> population that uses as chemotactic receptor Tar. If we use salicylate as chemoatracttant, the system will be composed by two bacterial strains: <i>Escherichia coli</i> (detection and signaling population) and <i>Pseudomonas putida G7</i> (chemotactic population), because the first one is not chemotactic to salicylate.
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<div class="center">
<div class="center">
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                  <a href="http://2010.igem.org/Team:UPO-Sevilla/Project/Chemotaxis"> <img class="subBanner" src="http://2010.igem.org/wiki/images/1/1f/BacterialCrowdingChemotaxisBanner.png" alt="Chemotaxis" /></a>
 
                   <a href="http://2010.igem.org/Team:UPO-Sevilla/Project/Sensing"><img class="subBanner" src="http://2010.igem.org/wiki/images/f/f8/BacterialCrowdingSensingBanner.png" alt="Sensing" /></a>
                   <a href="http://2010.igem.org/Team:UPO-Sevilla/Project/Sensing"><img class="subBanner" src="http://2010.igem.org/wiki/images/f/f8/BacterialCrowdingSensingBanner.png" alt="Sensing" /></a>
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                  <a href="http://2010.igem.org/Team:UPO-Sevilla/Project/Chemotaxis"> <img class="subBanner" src="http://2010.igem.org/wiki/images/1/1f/BacterialCrowdingChemotaxisBanner.png" alt="Chemotaxis" /></a>
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                   <a href="http://2010.igem.org/Team:UPO-Sevilla/Project/Assays"><img class="subBanner" src="http://2010.igem.org/wiki/images/0/0d/BacterialCrowdingAssaysBanner.png" alt="Assays" /></a>
                   <a href="http://2010.igem.org/Team:UPO-Sevilla/Project/Assays"><img class="subBanner" src="http://2010.igem.org/wiki/images/0/0d/BacterialCrowdingAssaysBanner.png" alt="Assays" /></a>
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                  <a href="http://2010.igem.org/Team:UPO-Sevilla/Project/Results"><img class="subBanner" src="http://2010.igem.org/wiki/images/d/dd/BacterialCrowdingResultsBanner.png" alt="Results" /></a>
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Latest revision as of 18:47, 27 October 2010

Abstract

The aim of our project is to concentrate a significant population of bacteria around a vegetable polysaccharidic surface, a non-difussible signal. This phenomenon was renamed as Bacterial Crowding. Therefore, it is necessary that the process of interaction with the surface by a few number of bacteria triggered the production and excretion of chemicals that, acting as chemoattractants, generate a chemical-diffusing gradient which could induce chemotactic process of close bacteria. These bacteria will redirect their random movements to the same plant surface and the concentration of the cell population will raise up in this region, thanks to an amplification process.

In order to get that effect it is necessary to use the Prh system, the only known sensing system able to detect non-diffusble signals. We also used the Fec system to design four signal transduction circuits which would finish with PrhI-dependent promoter PprhJ, which would activate specifically chemoattractant production (aspartate, glutamate or salicylate); or with FecI-dependent promoter PfecA, which would act in the same way. You can see these circuits here. However, we finally only focused on one signal transduction circuit which uses PrhA/FecA outer membrane fusion protein, performing an hybrid sensing system.

This project could use one or two bacterial strains, depending on the chemoattractant. If it is aspartate or glutamate it would be necessary only an E. coli population that uses as chemotactic receptor Tar. If we use salicylate as chemoatracttant, the system will be composed by two bacterial strains: Escherichia coli (detection and signaling population) and Pseudomonas putida G7 (chemotactic population), because the first one is not chemotactic to salicylate.

Assays Results
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