http://2010.igem.org/wiki/index.php?title=Special:Contributions/Dcabpra&feed=atom&limit=50&target=Dcabpra&year=&month=2010.igem.org - User contributions [en]2024-03-28T12:33:48ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:UPO-SevillaTeam:UPO-Sevilla2010-10-27T21:59:39Z<p>Dcabpra: </p>
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<h1>Team description</h1><br />
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<p>The UPO-Sevilla team was formed by the initiative of a group of Biotechnology students at the Universidad Pablo de Olavide, who heard about the iGEM contest and were interested in participating. Our team is currently formed by five Biotechnology students (Eva Fernández, David Caballero, Félix Reyes, Adrián Arellano and Paola Gallardo) and one Informatics student (Luis Eduardo Pavón), and is supervised by two professors, Luis Merino (Systems Informatics), and Fernando Govantes (Microbiology).</p><br />
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<img class="center" src="https://static.igem.org/mediawiki/2010/0/01/Sunflowers_pic_small.jpg" alt="UPO-Sevilla Team"/><br />
<p class="caption"><b>UPO-Sevilla team.</b> Left to right: Eva, Félix, David, Adrián, Fernan, Luis, Edu and Paola</p><br />
<br />
<h1>Project outline</h1><br />
<br />
<p>The possibility of specifically targeting bacteria to biological or abiotic surfaces is a promising technology of potential use in therapy, pest control and bioremediation, among others. However, since in most cases bacteria are not drawn towards their targets, the possibility of specific interaction is limited to those bacterial cells that randomly collide with the surface, thus requiring a high bacterial population to achieve efficient targeting. On the other hand, most bacteria are specifically attracted by gradients of a variety of chemicals, thus achieving high cell densities in the areas where the chemoattractants are present at higher concentration. The aim of this project is to explore the possibility of directing a relatively small population of bacteria to interact efficiently with a non-diffusible target exposed on a biotic or abiotic surface. To achieve this goal, our proposal involves the construction of bacterial strains harboring a two-tiered regulatory circuit involving the following elements:</p><br />
<br />
<ol><br />
<li>A cell surface-signaling circuit based on the Prh system of Ralstonia solanacearum to detect surface-associated polysaccharides and activate gene expresion. The Prh system has the unique property of detecting a non-diffusible signal present in plant cell polysaccharides and transducing that signal across the outer membrane, the periplasm and the cell membrane to activate transcription by means of the alternative extracytoplasmic function sigma factor PrhI. We will also make use of the Fec system of E. coli, which is very similar to Prh system. This way, using a fusion protein of PrhA-FecA, we will connect this two systems, Prh and Fec, and as a result it is expected to interact with FecR, and activate FecI. </li> <br />
<li>A gene expression system based on the FecI-dependent promoter P<i>fecA</i> engineered to activate synthesis and excretion of a suitable chemoatractant (CA) for the host bacteria upon interaction with surface-exposed polysaccharides.<br />
The expected response is the accumulation of bacterial cells on the polysaccharide-loaded surface, as the initial attachment of a few cells will be amplified by the secondary chemotactic response triggered by the interaction with the surface (Figure 1).</li><br />
</ol><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/3/39/Figura_crowding.jpg" alt="UPO-Sevilla Team"/><br />
<p class="caption"><b>Figure 1. Basic elements of the project</b>. Detection of specific polysaccharides on the plant cell wall (yellow stars) by random collision with the plant cells is transduced by the Prh system to activate the synthesis of a CA that is excreted to the medium. The concentration gradient of the CA directs additional bacteria to the vicinity of the plant cell, thus increasing the likelihood of interaction with the specific targets. This results in the synthesis of more CA and the amplification of the response. Eventually, a large population of bacterial cells accumulates on the surface of the plant cell wall.</p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-SevillaTeam:UPO-Sevilla2010-10-27T21:58:18Z<p>Dcabpra: </p>
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<h1>Team description</h1><br />
<br />
<p>The UPO-Sevilla team was formed by the initiative of a group of Biotechnology students at the Universidad Pablo de Olavide, who heard about the iGEM contest and were interested in participating. Our team is currently formed by five Biotechnology students (Eva Fernández, David Caballero, Félix Reyes, Adrián Arellano and Paola Gallardo) and one Informatics student (Luis Eduardo Pavón), and is supervised by two professors, Luis Merino (Systems Informatics), and Fernando Govantes (Microbiology).</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/0/01/Sunflowers_pic_small.jpg" alt="UPO-Sevilla Team"/><br />
<p class="caption"><b>UPO-Sevilla team.</b> Left to right: Eva, Félix, David, Adrián, Fernan, Luis, Edu and Paola</p><br />
<br />
<h1>Project outline</h1><br />
<br />
<p>The possibility of specifically targeting bacteria to biological or abiotic surfaces is a promising technology of potential use in therapy, pest control and bioremediation, among others. However, since in most cases bacteria are not drawn towards their targets, the possibility of specific interaction is limited to those bacterial cells that randomly collide with the surface, thus requiring a high bacterial population to achieve efficient targeting. On the other hand, most bacteria are specifically attracted by gradients of a variety of chemicals, thus achieving high cell densities in the areas where the chemoattractants are present at higher concentration. The aim of this project is to explore the possibility of directing a relatively small population of bacteria to interact efficiently with a non-diffusible target exposed on a biotic or abiotic surface. To achieve this goal, our proposal involves the construction of bacterial strains harboring a two-tiered regulatory circuit involving the following elements:</p><br />
<br />
<ol><br />
<li>A cell surface-signaling circuit based on the Prh system of Ralstonia solanacearum to detect surface-associated polysaccharides and activate gene expresion. The Prh system has the unique property of detecting a non-diffusible signal present in plant cell polysaccharides and transducing that signal across the outer membrane, the periplasm and the cell membrane to activate transcription by means of the alternative extracytoplasmic function sigma factor PrhI. We will also make use of the Fec system of E. coli, which is very similar to Prh system. This way, using a fusion protein of PrhA-FecA, we will connect this two systems, Prh and Fec, and as a result it is expected to interact with FecR, and activate FecI. </li> <br />
<li>A gene expression system based on the FecI-dependent promoter P<i>fecA</i> engineered to activate synthesis and excretion of a suitable chemoatractant (CA) for the host bacteria upon interaction with surface-exposed polysaccharides.<br />
The expected response is the accumulation of bacterial cells on the polysaccharide-loaded surface, as the initial attachment of a few cells will be amplified by the secondary chemotactic response triggered by the interaction with the surface (Figure 1).</li><br />
</ol><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/3/39/Figura_crowding.jpg" alt="UPO-Sevilla Team"/><br />
<p class="caption"><b>Figure 1. Basic elements of the project</b>. Detection of specific polysaccharides on the plant cell wall (yellow stars) by random collision with the plant cells is transduced by the Prh system to activate the synthesis of a CA that is excreted to the medium. The concentration gradient of the CA directs additional bacteria to the vicinity of the plant cell, thus increasing the likelihood of interaction with the specific targets. This results in the synthesis of more CA and the amplification of the response. Eventually, a large population of bacterial cells accumulates on the surface of the plant cell wall.</p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/ProjectTeam:UPO-Sevilla/Project2010-10-27T18:47:59Z<p>Dcabpra: </p>
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<h1>Abstract</h1><br />
<br />
<br />
<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><br />
<br />
<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="https://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><br />
<br />
<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.<br />
</p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Project/AbstractTeam:UPO-Sevilla/Project/Abstract2010-10-27T18:47:34Z<p>Dcabpra: </p>
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<h1>Abstract</h1><br />
<br />
<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><br />
<br />
<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="https://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><br />
<br />
<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.<br />
</p><br />
<br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_19Team:UPO-Sevilla/Notebook/10 192010-10-27T15:44:12Z<p>Dcabpra: </p>
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<h1>October, 19th</h1><br />
<h2>Assay Team</h2><br />
<p>We adjusted O.D. of cultures to the proper one, prepared chambers and capillaries with the different media(aspartate or motility buffer) and checked assays at 30ºC and at RT. We took microscopy images every 15 minutes(till 60') in the entrance of the capillaries. Also, we emptied the content of capillaries in Eppendorfs containing 200ul of motility buffer. We spread dilutions on LB+Ap plates.</p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_19Team:UPO-Sevilla/Notebook/10 192010-10-27T15:43:32Z<p>Dcabpra: </p>
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<h1>October, 19th</h1><br />
<h2>Assay Team</h2><br />
<p>We adjusted the D.O. to the proper one, prepared chambers and capillaries with the different media(aspartate or motility buffer) and checked assays at 30ºC and at RT. We took microscopy images every 15 minutes(till 60') in the entrance of the capillaries. Also, we emptied the content of capillaries in Eppendorfs containing 200ul of motility buffer. We spread dilutions on LB+Ap plates.</p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_15Team:UPO-Sevilla/Notebook/10 152010-10-27T15:37:50Z<p>Dcabpra: </p>
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<h1>October, 15th</h1><br />
<br />
<h2>Assay Team</h2><br />
<br />
<p>Today we have prepared new chemotaxis assays: Adler capillary assay, Adler capillary assay in 96-well PVC microplates and capillary assay using syringe and needles’ cap. The aim is to carry out a good chemotaxis assay definitely.</p><br />
<p>We have set up inocula of <i>E. coli</i>RP437 and RP6665 in minimal medium with glycerol and supplements at 30º in low shaking overnight. </p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_13Team:UPO-Sevilla/Notebook/10 132010-10-27T15:36:42Z<p>Dcabpra: </p>
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<h1>October, 13th</h1><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>Inocula of 12+2+13+3 different candidates were set up.</p><br />
<p>Minipreps of 12+2+16+3 different inocula and digestion of them (2h, 37ºC). Samples were run an agarose gel. We have positive preparations.<br />
</p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_12Team:UPO-Sevilla/Notebook/10 122010-10-27T15:36:08Z<p>Dcabpra: </p>
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<h1>October, 12th</h1><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>Transformation of UPO 12+2+13+3.</p><br />
<p>Inocula of 12+2+16+3 different colonies were set up.<br />
</p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_09Team:UPO-Sevilla/Notebook/10 092010-10-27T15:35:12Z<p>Dcabpra: </p>
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<h1>October, 9th</h1><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>UPO 4, 5 and 11 were run in an agarose gel. UPO 4 and 5 were confirmed. Inocula of different candidates of UPO 11 were set up.</p><br />
<p>Inocula of 13+3/3T5 different colonies were set up.</p><br />
<p>Transformation of 1+2+7+3 ligations in DH5α.</p><br />
<p>Ligation of UPO 12+2+16+3 (3A and SA).<br />
</p><br />
<br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_08Team:UPO-Sevilla/Notebook/10 082010-10-27T15:34:50Z<p>Dcabpra: </p>
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<h1>October, 8th</h1><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>Minipreps of 16+3 different inocula and digestion of them (2h, 37ºC). Samples were run in a 0.8% agarose gel. UPO16+3/3T5 was confirmed.</p><br />
<p>Minipreps and digestion of UPO4, 5 and 11/1C3 inocula (2h, 37ºC). Samples were run in a 0.8% agarose gel. We had to repeat these digestions because there were little DNA quantity. Digestion again.</p><br />
<p>Transformation of 13+3/3T5 ligations in DH5α.</p><br />
<p>Digestion of UPO 1+2 and 7+3 (2h, 37ºC). Preparations were run in a 0.8% agarose gel and purified by using GFX. Ligation of 1+2+7+3. </p><br />
<p>12+2 primers pair were denatured and renatured. UPO 16+3 was digested (to 3A and SA) and checked by electrophoresis gel. Spots were isolated and purified using GFX.<br />
</p><br />
<br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_06Team:UPO-Sevilla/Notebook/10 062010-10-27T15:34:25Z<p>Dcabpra: </p>
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<h1>October, 6th</h1><br />
<br />
<h2>AssemblyTeam</h2><br />
<br />
<p>Minipreps of 13+3 different inocula and digestion of them (2h, 37ºC). Samples were run in a 0.8% agarose gel. </p><br />
<p>Transformation of 16+3/3T5 ligations in DH5α.</p><br />
<p>Transformation of UPO4, 5 and 11/1C3 ligations in DH5α.</p><br />
<br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_14Team:UPO-Sevilla/Notebook/10 142010-10-27T15:33:00Z<p>Dcabpra: </p>
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<h1>October, 14th</h1><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>Minipreps of 12+2+13+3 different inocula and digestion of them (2h, 37ºC). Samples were run in a 0.8% agarose gel. UPO 12+2+13+3 was confirmed. </p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_13Team:UPO-Sevilla/Notebook/10 132010-10-27T15:32:22Z<p>Dcabpra: </p>
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<h1>October, 13th</h1><br />
<br />
<h2>Assay Team</h2><br />
<br />
<p>Inocula of 12+2+13+3 different candidates were set up.</p><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>Minipreps of 12+2+16+3 different inocula and digestion of them (2h, 37ºC). Samples were run an agarose gel. We have positive preparations.<br />
</p><br />
<br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_11Team:UPO-Sevilla/Notebook/10 112010-10-27T15:28:54Z<p>Dcabpra: </p>
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<h1>October, 11th</h1><br />
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<h2>Assembly Team</h2><br />
<br />
<p>Minipreps of 1+2+7+3 different inocula and digestion of them (2h, 37ºC). 22 samples were run in an agarose gel. Results were negative. This device has a problem: either it is not viable or 1+2 quantity is not enough. Other UPO 1+2 preparation was made but using more DNA.</p><br />
<p>Ligation of 12+2+13+3 (3A and SA).<br />
</p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_10Team:UPO-Sevilla/Notebook/10 102010-10-27T15:27:56Z<p>Dcabpra: </p>
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<h1>October, 10th</h1><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>Minipreps of UPO 11 samples and digestion. UPO 11 was confirmed by agarose gel. </p><br />
<p>Minipreps of 13+3 different inocula and digestion of them (2h, 37ºC). Samples were run in a 0.8% agarose gel. UPO13+3/3T5 was confirmed. </p><br />
<p>Inocula of 1+2+7+3 different colonies were set up.</p><br />
<p>Transformation of UPO 12+2+16+3.</p><br />
<p>Digestion of 13+3 (to 3A and SA).<br />
</p><br />
<br />
<br />
<a class="return_button" href="/Team:UPO-Sevilla/Notebook/10_09" title="Go to 9th of October"><span>Go to 9th of October</span></a><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_09Team:UPO-Sevilla/Notebook/10 092010-10-27T15:27:18Z<p>Dcabpra: </p>
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<h1>October, 9th</h1><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>UPO 4, 5 and 11 were run in an agarose gel. UPO 4 and 5 were confirmed. Inocula of different candidates of UPO 11 were set up.</p><br />
<p>Inocula of 13+3/3T5 different colonies were set up.</p><br />
<p>Transformation of 1+2+7+3 ligations in DHTα.</p><br />
<p>Ligation of UPO 12+2+16+3 (3A and SA).<br />
</p><br />
<br />
<br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_08Team:UPO-Sevilla/Notebook/10 082010-10-27T15:25:22Z<p>Dcabpra: </p>
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<h1>October, 8th</h1><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>Minipreps of 16+3 different inocula and digestion of them (2h, 37ºC). Samples were run in a 0.8% agarose gel. UPO16+3/3T5 was confirmed.</p><br />
<p>Minipreps and digestion of UPO4, 5 and 11/1C3 inocula (2h, 37ºC). Samples were run in a 0.8% agarose gel. We had to repeat these digestions because there were little DNA quantity. Digestion again.</p><br />
<p>Transformation of 13+3/3T5 ligations in DHTα.</p><br />
<p>Digestion of UPO 1+2 and 7+3 (2h, 37ºC). Preparations were run in a 0.8% agarose gel and purified by using GFX. Ligation of 1+2+7+3. </p><br />
<p>12+2 primers pair were denatured and renatured. UPO 16+3 was digested (to 3A and SA) and checked by electrophoresis gel. Spots were isolated and purified using GFX.<br />
</p><br />
<br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_06Team:UPO-Sevilla/Notebook/10 062010-10-27T15:17:15Z<p>Dcabpra: </p>
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<h1>October, 6th</h1><br />
<br />
<h2>AssemblyTeam</h2><br />
<br />
<p>Minipreps of 13+3 different inocula and digestion of them (2h, 37ºC). Samples were run in a 0.8% agarose gel. </p><br />
<p>Transformation of 16+3/3T5 ligations in DHTα.</p><br />
<p>Transformation of UPO4, 5 and 11/1C3 ligations in DHTα.</p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_05Team:UPO-Sevilla/Notebook/10 052010-10-27T15:14:24Z<p>Dcabpra: </p>
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<h1>October, 5th</h1><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>We have set up inocula of 13+3 different colonies.</p><br />
<p>Digestion of UPO 16+3 (2h, 37ºC) to clone them at pSB3T5 (final vector of 12+2+16+3 circuit). Digestion was run in a 0.8% agarose gel and the positive spot was isolated and purified using GFX. The purification was quantified by Nanodrop. Ligation of 16+3/3T5. </p><br />
<p>Digestion of UPO4, UPO5 and UPO11 (2h, 37ºC) to clone them at pSB1C3. Digestions were run in an agarose gel and positive spots were isolated and purified using GFX. Ligation of UPO4, 5 and 11/1C3.</p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_02Team:UPO-Sevilla/Notebook/10 022010-10-27T15:12:53Z<p>Dcabpra: </p>
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<h1>October, 2nd</h1><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>Colony PCR of positive candidates from our transformation plates. We had positive results of 7, 7+3 and 16+3/4K5.</p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/10_01Team:UPO-Sevilla/Notebook/10 012010-10-27T15:12:19Z<p>Dcabpra: </p>
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<h1>October, 1st</h1><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>Different samples were run in a 0.8% or 1.5% agarose gel. UPO13+3, 12+2 and 1+2+16+3 were confirmed.</p><br />
<br />
<p>We have checked different preparations of 1+2 and 12+2. Different samples of 1+2 were digested using SspI-SpeI and these digestions were run in a 1.5% agarose gel. PCRs of 12+2 (several versions) and 12 were made and were run in a 0.8% polyacrilamide/TBE gel. UPO1+2 was confirmed; but UPO12+2 results were negative. UPO12+2 primers were designed.</p><br />
<br />
<p>Digestion of UPO 7, 7+3, 13+3 and 16+3 (2h, 37ºC) to clone them at pSB4K5. Digestions were run in an agarose gel and positive spots (all of them except 13+3) were isolated and purified using GFX. Ligation and transformation in DH5α.</p><br />
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<h1>September, 7th</h1><br />
<br />
<h2>Production Team</h2><br />
<br />
<p> We performed the first overlapping PCR for SDM. We performed three reactions to be able of obtain <i>fecI-fecR</i> and <i>fecR</i> parts by mutagenesis. In each reaction the mix was:</p><br />
<br />
<p>37’5 ul H2O + 0,5ul UPO28* + 4 ul dNTP (10mM) + 5 ul Pfu buffer + 1 ul Pfu + 1 ul A-primer + 1 ul B-primer = 50 ul Vt</p><br />
<br />
<p>Next we performed a preparative electrophoresis with PCR’s products. 0,8% gel electrophoresis showed that we obtained amplification enough to mutagenize <i>fecR</i>, but not <i>fecI-fecR</i> composite part. We isolated from gel the amplified parts, products of the first PCR reaction needed to the next one.</p><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>We analyzed our work and determined the most important biobricks to be built. Most of them are still uncompleted, but time is running out. A new distribution of the sources was done this week. We began with the digestion of the unavailable biobricks and the purification of available ones. We lost 2+6 (not digested). After that, we proceeded to join the biobricks: (1+2)+(7+2), 4+3, 4+ pSB1C3 by ligation (o/n)</p><br />
<br />
<h2>Assay Team</h2><br />
<br />
<p>We started with a chemotaxis assays series today. We studied some possible strategies we could follow in order to see chemotaxis in bacteria (quantitative and qualitative assays).</p><br />
<br />
<p>We designed the method to put into practice the capillary assay developed by Adler and we prepared the material to carry it out.</p><br />
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<h1>September, 3rd</h1><br />
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<br />
<br />
<p>We had the opportunity to show our project to John S. Parkinson before a seminar he gave in Sevilla. Thanks his ideas we were nearer of successful chemotaxis assays.</p><br />
<br />
<p>Besides he confirmed our expectation that glutamate is a worse attractant than aspartate for <i>E. coli</i>. Furthermore he told us that <i>E. coli</i> is able to exudates aspartate. A lot of useful information.</p><br />
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<h1>September, 01st</h1><br />
<br />
<h2>Production Team</h2><br />
<br />
<p>We wrote the protocol for <i>fecI-fecR*</i> (UPO28*) SDM. It was necessary to remove a PstI site in 1184<sup>th</sup>pb (inside of <i>fecR*</i> secuence). We were going to perform two strategies:</p><br />
<br />
<ul><br />
<li><i>fecI-fecR*</i> SDM to get a composed part.</li><br />
<br />
<li><i>fecR*</i> SDM to get only a fecR part. We already had fecI part.</li><br />
</ul><br />
<br />
<p>It was not necessary to mutagenize <i>fecA*</i> (UPO8*) or <i>gltD**</i> (UPO17) since we were not going to keep on building devices of them.</p><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>We analyzed all the information we had about parts and devices and took a decision: we were going to do without device 2 (Fec system) and devices 10, 11 and 12 (Glutamate Synthase). The explication was:</p><br />
<br />
<ul><br />
<li>D2 was a control of the sensing system. Moreover we could not synthesize fecA part.</li><br />
<br />
<li>D10, D11 and D12: glutamate synthase subunits were pretty big so they could give us problems. Also we had problems to remove EcoRI target in one of them by SDM. It was not a huge deal not to continue with that chemoattractant synthesize system because we had other two: aspartate to <i>E. coli</i> and salicilate to <i>P. putida.</i></li><br />
</ul><br />
<br />
<p>We wrote protocols for next devices, SDM of UPO28 and digestion analysis of 1+19 and 16+3.</p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/08_13Team:UPO-Sevilla/Notebook/08 132010-10-27T14:59:09Z<p>Dcabpra: </p>
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<h1>August, 13th</h1><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>There were kept inocula of verified devices to save them at -80ºC. We made plasmid purifications by using those inocula and we analyzed them again by EcoRI and PstI cutting and agarose gel electrophoresis. Sadly, not all the analysis showed positive results. Then we made other analytic digestions to check those devices and the day before devices.</p><br />
<br />
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<thead><br />
<tr><br />
<th>PARTS</th><br />
<th>RESTRICTION ENZYMES</th><br />
<th>PART SIZES</th><br />
<th>FRAGMENT SIZES</th><br />
<th>RESULTS</th><br />
</tr><br />
</thead><br />
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<tr><br />
<td>UPO2</td><br />
<td>SspI + PstI</td><br />
<td>15</td><br />
<td>1892, 271</td><br />
<td>&#10004;</td><br />
</tr><br />
<tr><br />
<td>1+2</td><br />
<td>SspI + PstI</td><br />
<td>35+15</td><br />
<td>1892, 315</td><br />
<td>&#10004;</td><br />
</tr><br />
<tr><br />
<td>pSB1A3 (control)</td><br />
<td>SspI + PstI</td><br />
<td>2157</td><br />
<td>1912, 340+1200</td><br />
<td>&#10004;</td><br />
</tr><br />
<tr><br />
<td>UPO12 (pMA)</td><br />
<td>PstI + NruI</td><br />
<td>86</td><br />
<td>it would be just one cut</td><br />
<td>&#10004;</td><br />
</tr><br />
<tr><br />
<td>12+2</td><br />
<td>PstI + NruI</td><br />
<td>86+15</td><br />
<td>1995, 589</td><br />
<td>&#10005;</td><br />
</tr><br />
<tr><br />
<td>pSB1T3 (control)</td><br />
<td>PstI + NruI</td><br />
<td>2416</td><br />
<td>1995, 488+1200</td><br />
<td>&#10004;</td><br />
</tr><br />
<tr><br />
<td>UPO19 (1AK3)</td><br />
<td>EcoRI + PvuII</td><br />
<td>1892</td><br />
<td>1077, 815+3189</td><br />
<td>&#10004;</td><br />
</tr><br />
<tr><br />
<td>1+19 (1T3)</td><br />
<td>EcoRI + PvuII</td><br />
<td>35+1892</td><br />
<td>1112, 815+2463</td><br />
<td>?</td><br />
</tr><br />
<tr><br />
<td>UPO19 (1AK3)</td><br />
<td>PvuII + PstI</td><br />
<td>1892</td><br />
<td>815, 1077+3189</td><br />
<td>&#10004;</td><br />
</tr><br />
<tr><br />
<td>1+19 (1T3)</td><br />
<td>PvuII + PstI</td><br />
<td>35+1892</td><br />
<td>815, 1112+2463</td><br />
<td>&#10004;</td><br />
</tr><br />
<tr><br />
<td>11+2+16+3 (3C5)</td><br />
<td>EcoRI + BglII</td><br />
<td>192+15+1518+129</td><br />
<td>279, 1239+V</td><br />
<td>&#10005;</td><br />
</tr><br />
<tr><br />
<td>12+2+16+3 (3C5)</td><br />
<td>EcoRI + BglII</td><br />
<td>86+15+1518+129</td><br />
<td>173, 1239+V</td><br />
<td>&#10005;</td><br />
</tr><br />
<tr><br />
<td>16+3 (1C3)</td><br />
<td>EcoRI + BglII</td><br />
<td>1518+129</td><br />
<td>72, 1239+V</td><br />
<td>?</td><br />
</tr><br />
<tr><br />
<td>2+4+2+6</td><br />
<td>pending</td><br />
<td>pending</td><br />
<td>pending</td><br />
<td>&#10005;</td><br />
</tr><br />
<tr><br />
<td>1+2+4</td><br />
<td>EcoRI + SphI</td><br />
<td>35+15+2316</td><br />
<td>716, 1650+V</td><br />
<td>&#10005;</td><br />
</tr><br />
<tr><br />
<td>2+4</td><br />
<td>EcoRI + SphI</td><br />
<td>35+15+2316</td><br />
<td>661, 1650+V</td><br />
<td>&#10005;</td><br />
</tr><br />
<tr><br />
<td>1+2+7+2</td><br />
<td>EcoRI + SspI</td><br />
<td>35+15+2292+15</td><br />
<td>161, 2181+V</td><br />
<td>&#10005;</td><br />
</tr><br />
<tr><br />
<td>7+2</td><br />
<td>EcoRI + SspI</td><br />
<td>2292+15</td><br />
<td>111 ,2181+V</td><br />
<td>&#10004;</td><br />
</tr><br />
<tr><br />
<td>11+19</td><br />
<td>EcoRI + EcoRV</td><br />
<td>192 + 1892</td><br />
<td>280, 1804+V</td><br />
<td>&#10005;</td><br />
</tr><br />
<tr><br />
<td>19</td><br />
<td>&nbsp;</td><br />
<td>1892</td><br />
<td>88, 1804+V</td><br />
<td>&#10004;</td><br />
</tr><br />
<tr><br />
<td>12+2+13+3</td><br />
<td>EcoRI + NcoI</td><br />
<td>86+15+720+129</td><br />
<td>216, 554+V</td><br />
<td>&#10005;</td><br />
</tr><br />
<tr><br />
<td>13+3</td><br />
<td>EcoRI + NcoI</td><br />
<td>720+129</td><br />
<td>166, 554+V</td><br />
<td>&#10005;</td><br />
</tr><br />
<tr><br />
<td>UPO28</td><br />
<td>SphI + PstI</td><br />
<td>1472</td><br />
<td>1121, 351+V</td><br />
<td>&#10004;</td><br />
</tr><br />
<tr><br />
<td>UPO28</td><br />
<td>SphI + EcoRI</td><br />
<td>1472</td><br />
<td>351, 1121+V</td><br />
<td>&#10005;</td><br />
</tr><br />
<tr><br />
<td>2+18</td><br />
<td>EcoRI +SphI</td><br />
<td>pending</td><br />
<td>pending</td><br />
<td>pending</td><br />
</tr><br />
<tr><br />
<td>18</td><br />
<td>EcoRI +SphI</td><br />
<td>pending</td><br />
<td>pending</td><br />
<td>pending</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<br />
<p>We had three kinds of results:</p><br />
<br />
<ul><br />
<li>Confirmed devices: 1+2, 7+2, 13+3.</li><br />
<br />
<li>Not assembled devices: 12+2, 2+4, 28, 11+19, 1+2+4, 11+2+16+3, 12+2+16+3, 1+2+7+2, 12+2+13+3.</li><br />
<br />
<li>Need another analysis: 1+19, 16+3.</li><br />
</ul><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/08_12Team:UPO-Sevilla/Notebook/08 122010-10-27T14:57:33Z<p>Dcabpra: </p>
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<h1>August, 12th</h1><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>We made plasmid minipreps and analytic digestions with EcoRI and PstI by using inocula that we set up the day before. Digestion analysis was not conclusive since agarose gel ran out bad and we could not analyze it well. We were going to make analytic cuts by using restriction enzyme target which allowed us to distinguish the different devices.</p><br />
<br />
<p>Minipreps and analytic digestions of UPO 5 & UPO11 had been confirmed.</p><br />
<br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/08_11Team:UPO-Sevilla/Notebook/08 112010-10-27T14:55:38Z<p>Dcabpra: </p>
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<h1>August, 11th</h1><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>We had a lot of red and white colonies in every plate; except on: 12+19, 11+2+13+3 and 1+2+16+3 plates. There were no colonies on control plates. We made colony PCR reactions for six colonies from each plate. Agarose gel analysis showed that running time was too long. We could not rule out anything, so we set up inocula of each candidate.</p><br />
<br />
<p>We set up inocula of positive candidate from UPO 4, UPO 5 and UPO 11 plates. We made minipreps and analytic digestions with the day before inocula. 0.8% agarosa gel analysis confirmed 2+18 and 2+4+2+6.</p><br />
<br />
<p>We made colony PCR by using colonies from 18+3 and 2+6+2+5+3 plates: no positive results.</p><br />
<br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/08_09Team:UPO-Sevilla/Notebook/08 092010-10-27T14:52:55Z<p>Dcabpra: </p>
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<h1>August, 9th</h1><br />
<br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>We analyzed plaques spread the day before; it looked like if the bacteria could not grow. There were only three red colonies (negatives) in a plaque. Those devices had promoter and were assembled in high copy vectors. This is why we thought that the high expression of the devices could be harmful for bacteria. We analyzed the digestions in agarose gel to verify it. Almost all the digestions were well made and we only had to repeat two. In order to avoid that fact again, we assembled devices including promoters in low copy vectors. Like in before cases, we used different vectors depending of the resistance of the origin vectors we need. Below it is summed up:</p><br />
<br />
<ul><br />
<li>pSB4K5: (1+2) + (4)</li><br />
<br />
<li>pSB4C5: (1+2) + (7+2)</li><br />
<br />
<li>pSB3T5: (11) + (19) // (12) + (19)</li><br />
<br />
<li>pSB3C5: (11+2) + (13+3) // (12+2) + (13+3) // (11+2) + (16+3) // (12+2) + (16+3) // (1+2) + (16+3)</li><br />
</ul><br />
<br />
<p>It was made the necessary digestions and we left ligation over night at 13ºC on average.</p><br />
<br />
<p>In other hand, digestion: 2+5+3 with BamI-PstI. We started the following devices (3A method): 7+2+28; 2+6+2+5+3; 2+18; 28+3; 2+4+2+6; 2+28; 18+3: digestion, ligation and transformation.</p><br />
<br />
<h2>DryLab Team</h2><br />
<br />
<p><strong>Modeling: </strong></p><br />
<br />
<p>Simulation issues concerning concentrations' stability fixed. Concentrations behave as expected.</p><br />
<br />
<p>Beginning of <i>E-coli</i> movement modeling, basing on tumbling and straight swim. </p><br />
<br />
<p>First implementation of a random movement is successful. </p><br />
<br />
<p>Simulation of bacterial movement in an environment with Chemoattractants started. Bacteria move towards the concentration gradient, but they tend to move futher away once they reach the highest concentration point. Besides, when bacteria approach a side of the recipient they are in, they usually experience troubles coming back.</p><br />
<br />
<p>By the end of the day, the aforementioned issues are all fixed.</p><br />
<br />
<br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/08_06Team:UPO-Sevilla/Notebook/08 062010-10-27T14:50:33Z<p>Dcabpra: </p>
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<h1>August, 6th</h1><br />
<br />
<h2>Production Team</h2><br />
<br />
<p>Minipreps were prepared from inocula. We digested with EcoRI and PstI for 2 hours at 37ºC. We ran an electrophoresis of the digestion products and we observed 1'5 kb fragments. We've got fecI-fecR. Colony PCR seems not to be a reliable proof.</p><br />
<br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>We used 3A method to build: 1+2+7+2, 11+2+13+3, 12+2+13+3, 11+2+16+3 and 12+2+16+3. The necessary steps were: digestion with accurate digestion enzyme, ligation and transformation. Plates were incubated at 37º over night.</p><br />
<br />
<p>Minpreps using inocula we set up the day before. Analytic digestion confirmed the next biobriks: UPO6, UPO7, UPO2+6, UPO12+2 and UPO11+2.</p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/08_05Team:UPO-Sevilla/Notebook/08 052010-10-27T14:49:00Z<p>Dcabpra: </p>
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<h1>August, 5th</h1><br />
<br />
<h2>Production Team</h2><br />
<br />
<p><strong>Paola Gallardo.</strong> We only found white colonies in fecI-fecR plates, and no colonies in control plates. We made colony PCR reactions to test five colonies, but we got negative results. ¿Why? We used lineal vectors. It had no sense. We set up inocula of the same colonies.</p><br />
<br />
<p><strong>David Caballero.</strong> Going on with assembly team work, we purified plasmids with new devices. Next, plasmid minipreps were digested by using EcoRI and PstI enzymes. Agarose gel electrophoresis analysis showed some positive results: 12+2, 11+2, 7+2. Moreover UPO6 and UPO7 parts were included in pSB1C3 plasmid to send to the Registry of Standard Biological Parts. UPO6 and UPO7 were synthesized using MrGene services.</p><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>And again, and again, and again... </p><br />
<p>UPO 2+6 and 2+7 were analyzed by colony PCR. We have a positive candidate: 2+6! Mr.Gene biobricks digestions were checked: everything was ok.</p><br />
<p>UPO1+2+4 plate only had red colonies. This device was repeated: ligation and transformation. </p><br />
<p>Five possible positive candidates were grown on UPO2+5+3 plate: we set up inocula of these.</p><br />
<br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/08_04Team:UPO-Sevilla/Notebook/08 042010-10-27T14:46:20Z<p>Dcabpra: </p>
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<h1>August, 4th</h1><br />
<br />
<h2>Production Team</h2><br />
<br />
<p>Transformation of the ligation products in <i>E. coli</i> DH5-α and waited one night to<br />
see the hopeful colonies in plates.</p><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>Minipreps of 2+4 and 2+5 inocula. Analytic digestion of these. Digestion, ligation and transformation of 1+2+4, 2+5+3, 2+6 and 2+7.</p><br />
<br />
<br />
<h2>DryLab Team</h2><br />
<br />
<p><strong>Wiki: </strong></p><br />
<br />
<ul><br />
<li>Home section upgraded.</li><br />
<br />
<li>Teams division added to Notebook section.</li><br />
</ul><br />
<br />
<p><strong>Modeling: </strong> Beginning of chemotaxis simulation. First JAVA-based implementations about a substance's diffusion into an environment. Some problems encountered concerning concentrations' stability. </p><br />
<br />
<a class="return_button" href="/Team:UPO-Sevilla/Notebook/08_03" title="Go to 3rd of August"><span>Go to 3rd of August</span></a><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/08_03Team:UPO-Sevilla/Notebook/08 032010-10-27T14:45:24Z<p>Dcabpra: </p>
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<h1>August, 3rd</h1><br />
<br />
<h2>Production Team</h2><br />
<br />
<p>We digest <i>fecI-fecR</i> with PstI and EcoRI restriction enzymes , and run an electrophoresis. We purified from electrophoresis gel (1'5 pb) and ligate it with the vector C3L (chloramphenicol).</p><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>We analyzed several candidates from 2+4, 2+5 and 2+7 plates by colony PCR. We had positive candidates of UPO2+4 and UPO2+5. We set up inocula of these candidates and spread on LB+Tc plates.</p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/08_03Team:UPO-Sevilla/Notebook/08 032010-10-27T14:43:18Z<p>Dcabpra: </p>
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<h1>August, 3rd</h1><br />
<br />
<h2>Production Team</h2><br />
<br />
<p><strong>Paola Gallardo.</strong> We digest <i>fecI-fecR</i> with PstI and EcoRI restriction enzymes , and run an electrophoresis. We purified from electrophoresis gel (1'5 pb) and ligate it with the vector C3L (chloramphenicol).</p><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>We analyzed several candidates from 2+4, 2+5 and 2+7 plates by colony PCR. We had positive candidates of UPO2+4 and UPO2+5. We set up inocula of these candidates and spread on LB+Tc plates.</p><br />
<br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/07_30Team:UPO-Sevilla/Notebook/07 302010-10-27T14:41:47Z<p>Dcabpra: </p>
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<h1>July, 30th</h1><br />
<br />
<h2>Production Team</h2><br />
<br />
<p><strong>Paola Gallardo.</strong> We got white colonies in <i>gltD</i> plates (not too much) and red and white colonies (most of them were red) in <i>fecI-fecR</i> plates. We analyzed the colonies by PCR and electrophoresis (0'8 % agarose gel). Negatives results. At the same time, we made the second round of the PCR reactions for the SDM, but this time we used Taq polymerase. Positive results.</p><br />
<br />
<p><strong>David Caballero.</strong> We purified plasmids with <i>fecA*</i> inserts of the day before inocula. We digested plasmids and analyzed by 0.8% agarose gel electrophoresis. We got one positive, so we had a new part to make side-directed mutagenesis (SDM). Remember that <i>fecA*</i> had a PstI target which would be removed.</p><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>No colonies in transformation plates (UPO1+2).Ligation and transformation of devices:<br />
UPO12+UPO19; UPO13+UPO3 and UPO18+UPO3. Transformation of UPO1+UPO19. Colony PCR with bacteria which grew yesterday (UPO 12+2; UPO1+19 and UPO13+3). We set up inocula and spread in plate.</p><br />
<br />
<h2>DryLab Team</h2><br />
<br />
<p><strong>Wiki:</strong> First implementations of Notebook calendar worked on.</p><br />
<br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Project/ResultsTeam:UPO-Sevilla/Project/Results2010-10-27T14:40:20Z<p>Dcabpra: </p>
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<h1>Results</h1><br />
<br />
<h2>Building devices</h2><br />
<br />
<p>During this summer we tried to build a lot of devices (17 exactly) but we finally realized that they were too many for our first time. We had many problems assembling parts and checking the constructions: colony PCR did not work (or it just worked sometimes), digestion enzymes did not cut always, we could hardly obtain new parts by PCR or site-directed mutagenesis products, etc. We think most problems were caused because of the lab conditions. We worked in a practices lab and the material we used was not the best. When we noticed that the Jamboree date was too close, we had to focus in some devices and forget assembling others. <strong>Prioritizing</strong> some devices helped us to finish proposed devices. Our work moved forward faster.</p><br />
<br />
<p>You can see below the devices we finally built and what they are for.</p><br />
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<ul><br />
<li><strong>Device 6</strong>. Quantify induction of P<i>fecA</i> promoter by GFP fluorescence.</li><br />
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<img class="centerMedium" src="https://static.igem.org/mediawiki/2010/6/6a/BacterialCrowdingMiniDevice6.png" alt="Device 6 of Bacterial Crowding project"/><br />
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<li><strong>Device 8</strong>. Production of chemoattractant aspartate mediated by signal transduction Circuit 2 in response to ferric citrate (because we could not assemble Circuit 3).</li><br />
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<img class="centerMedium" src="https://static.igem.org/mediawiki/2010/c/c2/BacterialCrowdingMiniDevice8.png" alt="Device 8 of Bacterial Crowding project"/><br />
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<li><strong>Device 9</strong>. Constitutive production of chemoattractant aspartate.</li><br />
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<img class="centerMedium" src="https://static.igem.org/mediawiki/2010/5/58/BacterialCrowdingMiniDevice9.png" alt="Device 9 of Bacterial Crowding project"/><br />
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<li><strong>Device 14</strong>. Production of chemoattractant salicylate (for <i>P. putida</i>) mediated by signal transduction circuit 2 in response to ferric citrate (because we could not assemble Circuit 3).</li><br />
</ul><br />
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<img class="center" src="https://static.igem.org/mediawiki/2010/8/86/BacterialCrowdingMiniDevice14.png" alt="Device 14 of Bacterial Crowding project"/><br />
<br />
<p>As you can see, we could not assemble any device related to the <a href="https://2010.igem.org/Team:UPO-Sevilla/Project/Sensing" target="_blank">Sensing</a> part of the project. Until the last day we tried to assemble the device that codes FecA/PrhA outer membrane hybrid protein (Device 17) but we did not achieve it. It seemed that this protein could have some harmful effects in bacteria. We tried to express FecA/PrhA under the control of a middle-strong constitutive promoter. First we did that using a high copy vector, but we soon changed it to a low copy plasmid. Nevertheless we never obtained colonies that harvest the hybrid protein and could survive. Two days before Wiki freezing we looked up any inducible expression vector we could use instead of our constitutive promoter. We found <a href="https://2010.igem.org/Team:UPO-Sevilla/Project/Sensing" target="_blank">TetR repressed POP/RIPS generator</a>, which showed suitable features, but we did not have enough time to test it before the Jamboree date. Anyhow, we thought that a good way to solve outer membrane protein expression problems is to use inducible promoters.</p><br />
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<img class="centerMedium" src="https://static.igem.org/mediawiki/2010/c/c5/BacterialCrowdingMiniDevice17.png" alt="Device 17 of Bacterial Crowding project"/><br />
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<h2>Chemotaxis assays</h2><br />
<br />
<p>The main way to test our project was to use chemotaxis assays. Over August we started to read as much articles about chemotaxis assays as we found. We soaked of these processes and tested some of them. In <a href="https://2010.igem.org/Team:UPO-Sevilla/Project/Sensing" target="_blank">Assay</a> site you can read more about all the different kinds of experiments we performed. Also we modified some of this assays.</p><br />
<br />
<p>When we started we used three bacterial strains: <i>E. coli</i> K-12, <i>Pseudomonas sp</i>. and <i>Pseudomonas putida</i> G7; and three chemoattractans, glutamate, aspartate and salycilate. Little by little we narrowed the possibilities to <i>E. coli</i> K-12 as strain and <strong>aspartate</strong> as attractant, because of its high expected chemotactic response. However, we could not achieve good results in our chemotaxis assays using tip boxes as chemotaxis chambers and needles instead of capillaries. So we changed media conditions, needle thickness, we did different dilutions… but successful results did not arrive. Finally we realized the chance involved in our misfortune, our <i>E. coli</i> strain got a mutation which did not allow it to move or sense chemotaxis stimulus. We checked it by using a soft agarose plate assay.</p><br />
<br />
<p>Then we changed our strain for other known motile <i>E. coli</i> RP437 strain which came from <a href="http://chemotaxis.biology.utah.edu/Parkinson_Lab/people/people.html" target="_blank">Sandy Parkinson lab</a> (chemotaxis researcher). Using the new strain we performed <strong>lower scale assays</strong> (1&#956;l capillaries, no more that 1ml of bacterial suspensions), optimized some conditions and we achieved our goal. By microscopy and dilution and plating the chemotactic response of the <i>E. coli</i> towards aspartate was characterized. </p><br />
<br />
<p>Here some pictures of a capillary assay of <i>E. coli</i> chemotaxis toward aspartate are shown. As you can see while time passes more bacteria accumulate inside the capillary and arround it. This fact clearly supports the chemotaxis response of <i>E. coli</i>.</p><br />
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<img class="centerBig" src="https://static.igem.org/mediawiki/2010/6/6f/BacterialCrowdingCapillaryAssayMicroscope.png" alt="Capillary assay Pictures"/><br />
<p class="caption"><i> <strong> Results of a capillary assay using microscope techniques</strong>. We can see that the chemotatic response toward aspartate is increasing as time passes by. Also there are major differences between the control without aspartate and the control with aspartate.</i></p><br />
<br />
<p>In the last chemotaxis assay we performed before Wiki freezing we used as chemotaxis chamber a <strong>flow-chamber</strong> with three channels and <strong>1&#956;l glass capillaries</strong>. In both edges of each channel there was a capillary, one with aspartate and other filled with buffer. We carried out experiments with two chambers, one was incubated at room temperature during 1h and the other at 30ºC. After incubation capillaries were cleaned with water and their contents were diluted and spread in agar plates. Then plates were incubated overnight at 37ºC and counted. Below results of this assay are shown. The response to aspartate is much higher than to buffer and also the standard deviation is lower when the temperature is fixed at 30ºC.</p><br />
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<img class="centerBig" src="https://static.igem.org/mediawiki/2010/a/a1/BacterialCrowdingChemotaxisAssayResults.png" alt="Bacterial Crowding Chemotaxis Assay Results"/><br />
<br />
<h2>Producing bacteria</h2><br />
<br />
<p> The Bacterial Crowding project included sensing non-diffusible signals and <strong>producing</strong> a <strong>chemoattractant</strong> as response. In spite of the fact that we could not work with the sensing part of the project we wanted to test if our modified bacteria were able to produce enough chemoattractant to induce chemotactic behaviour in their partners. Although we had two different built chemoattractant production circuits, for salycilate and aspartate, we focused on the generation of aspartate to attract <i>E. coli</i>, due to its higher chemotactic response. Anyway, we started to do assays with supernatant quite late and results were not available to upload to the Wiki page before Jamboree date.</p><br />
<br />
<p>We designed several growing media in which the expression of the plasmids could be analyzed, mainly media to induce P<i>fecA</i> promoter. We set up inocula in these media and when they were saturated, we centrifugated and reject the pellets. The supernatants would be used in chemotaxis assays.</p><br />
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<h1>August, 2nd</h1><br />
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<h2>Production Team</h2><br />
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<p><strong>Paola Gallardo.</strong> We started a new round of PCR for SDM, but this time we prepared five samples of each one. We ran an electrophoresis to check the products and we had only got positive results for <i>fecI-fecR.</i></p><br />
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<p><strong>David Caballero.</strong> SDM is made by two PCR reactions. We performed the first PCR reactions for <i>fecA*,</i> <i>gltD**</i> and <i>fecI-fecR*</i> parts. <i>gltD**</i> had two restriction enzyme target. One of them was really near of start codon so we would get it out of there using a longer primer which overlapped the target site. After PCR reactions were performed, samples were running in electrophoresis gel. All samples were perfect. We purified samples from gel and performed the second SDM PCR reaction for each part. Results would be analyzed the next day.</p><br />
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<h2>Assembly Team</h2><br />
<br />
<p>Mr Gene biobricks have come! YIPPEE!!.</p><br />
<br />
<p>Today has been a hard day: On the one hand, we analyzed several devices using colony PCR (different candidates from the following plates: 12+2, 12+19, 18+3 and 1+19) and we had unsuccessful results. Probably, our PCR reactions were contaminated. On the other hand, some biobricks were digested, ligated and transformated (2+4, 2+5, 2+6, 2+7, 4+2, 5+2, 6+2, 7+2, 11+2, 2+3 and 11+19).</p><br />
<br />
<h2>DryLab Team</h2><br />
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<p><strong>Wiki: </strong>Notebook calendar completed. </p><br />
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<h1>July, 30th</h1><br />
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<h2>Production Team</h2><br />
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<p><strong>Paola Gallardo.</strong> We got white colonies in <i>gltD</i> plates (not too much) and red and white colonies (most of them were red) in <i>fecI-fecR</i> plates. We analyzed the colonies by PCR and electrophoresis (0'8 % agarose gel). Negatives results. At the same time, we made the second round of the PCR reactions for the SDM, but this time we used Taq polymerase. Positive results.</p><br />
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<p><strong>David Caballero.</strong> We purified plasmids with <i>fecA*</i> inserts of the day before inocula. We digested plasmids and analyzed by 0.8% agarose gel electrophoresis. We got one positive, so we had a new part to make side-directed mutagenesis (SDM). Remember that <i>fecA*</i> had a PstI target which would be removed.</p><br />
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<h2>Assembly Team</h2><br />
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<p>No colonies in transformation plates (UPO1+2).Ligation and transformation of devices:<br />
UPO12+UPO19; UPO13+UPO3 and UPO18+UPO3. Transformation of UPO1+UPO19. Colony PCR with bacteria which grew yesterday (UPO 12+2; UPO1+19 and UPO13+3). We put inocula and spread in a plate.</p><br />
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<h2>DryLab Team</h2><br />
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<p><strong>Wiki:</strong> First implementations of Notebook calendar worked on.</p><br />
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<h1>July, 29th</h1><br />
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<h2>Production Team</h2><br />
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<p><strong>Paola Gallardo.</strong> Transformation of the ligation products in <i>E. coli</i> DH5-α and waited one night to see the results.</p><br />
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<p><strong>David Caballero.</strong> In order to verify that our original sample of <i>fecA*</i>, amplified by PCR using <i>E. coli</i> genome like template, had not problem we analyzed it by 0.8% agarose gel electrophoresis: first <i>fecA*</i> PCR product and two digestion products of this one. Also we did colony PCR and electrophoresis analysis to some white colonies we found in the first plate in which we tried to transform <i>E. coli</i> with <i>fecA*</i>. The spot pattern showed that <i>fecA*</i> PCR product and its digestions were perfect. Moreover we had two candidate from colony PCR analysis. We set up inocula from them and isolated in plate to verify the next day.</p><br />
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<h2>Assembly Team</h2><br />
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<p>We make miniprep of some plasmids and vectors. Digestion and ligation of 1+19 and 18+3. Transformation of 1+19, 2+12, 12+19, 13+3.</p><br />
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<h2>DryLab Team</h2><br />
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<p><strong>Modeling:</strong> Attending to Continuous Systems and Simbilogy (MatLab) seminar set by Luis Merino.</p><br />
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<h1>July, 28th</h1><br />
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<h2>Production Team</h2><br />
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<p><strong>Paola Gallardo.</strong> We run an electrophoresis to check the result and we observed a lot of unspecific very intense bands, but the expected bands had few intensity. We isolated and purified the desired bands. We digested the products: <i>gltD</i> with XbaI and PstI, <i>fecI-fecR</i> with EcoRI and PstI for two hours. We joined them with different vectors and rested o/n.</p><br />
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<p><strong>David Caballero.</strong> <i>fecA*</i> inocula appeared in red color behind being incubating all night long. This meant that bacteria had plasmids with RFP (red fluorescent protein) instead of <i>fecA*</i>. So we could not use it. Checking <i>fecA*</i> plates we saw that colonies which come from our inocula were red two days after starting the incubation. We found a new white colony too, so we analyzed it by colony PCR and electrophoresis analysis, but the result was negative. Changing focus, we analyzed <i>gltB</i> and <i>fecI-fecR*</i> inocula: vector purification, restriction enzyme parts digestion and 0.8% agarose gel electrophoresis analysis. We had a positive result for <i>gltB</i> part and negative for <i>fecI-fecR*</i> part (not well digested). With this it had been tested a new synthesized part: <i>gltB.</i> The loss of <i>fecI-fecR*</i> is unimportant, since it had already been gained in another process made by a partner. By this time, we had got all searched parts by PCR saving <i>fecA*</i>. It was resisting.</p><br />
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<h2>Assembly Team</h2><br />
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<p>We have to test the UPO 1+2 candidate so we digest with Nhe1 to UPO1 and we can see the plasmid run into the gel like a lineal plasmid. Thus, finally we can say we have UPO 1+2.</p><br />
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<p>And again, we start with UPO 1+19 and UPO 12+2, 12+19, 13+3 y 1+2+16+3.</p><br />
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<h1>July, 27th</h1><br />
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<h2>Production Team</h2><br />
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<p><strong>Paola Gallardo.</strong> We made the second round of the PCR reaction for the SDM, using suitable primers and fragments (gltD1+ gltD2, fecI-fecR1 + fecI-fecR2), and changing the characteristics of the cycles. Purification of the obtained products.</p><br />
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<p><strong>David Caballero.</strong> Inocula analysis of <i>fecI</i> and P<i>fecA</i>: vector purification and 1.2% agarose gel electrophoresis analysis. We had positive results for both parts. Two tests have been made to verify the obtaining of these new parts we added to the biobrick catalog: colony PCR and digestive purify vector analysis. We set up inocula of parts <i>gltB</i> and <i>fecI-fecR*</i> to do the second testing the next day. On the other hand we performed colony PCR reactions to test three colonies emerged in <i>fecA*</i> transformed plate. 0.8% agarose gel electrophoresis showed multiple lines, some of them coincident with <i>fecA*</i> size (2.4 kbp). So we set up inocula of these three new candidates and let them incubate overnight shaking at 37ºC.</p><br />
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<h2>DryLab Team</h2><br />
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<p><strong>Wiki:</strong> Overall wiki design completed. Some details to be concluded.</p><br />
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<h1>July, 26th</h1><br />
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<h2>Production Team</h2><br />
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<p><strong>Paola Gallardo.</strong> Products of the first PCR reaction were analyzed by electrophoresis (0'8 %). We got four bands clearly-defined with the correct lengths (635bp and 732bp for <i>gltD</i>, 1206bp and 312bp for <i>fecI-fecR</i>). Purification from gel.</p><br />
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<p><strong>David Caballero.</strong> Plate analysis: colonies were presented in all plates except for <i>fecA*</i> and control plates. We verified the presence of <i>fecI</i> and P<i>fecA</i> parts in colonies by colony PCR reactions and 1.5% agarose gel electrophoresis analysis. In other way, to obtain <i>fecA*</i> part we digested by restriction enzymes the amplified by PCR part again, ligated it and transformed competent bacteria which remained overnight at 37ºC in LB+Km plate.</p><br />
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<h2>Assembly Team</h2><br />
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<p>We repeated PCR colony reaction because we hadn’t anything confirmed yet. We analyzed UPO1+UPO2 with Nhe1 but results were negative. Moreover, we did PCR colony reaction for the new UPO1+2 part. We are moving forward too slow. In addition we are waiting for the synthesized DNA... We hope it comes soon!</p><br />
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<p>And again, digestion and ligation of UPO1 and UPO2 using the GINGO kit. Later, transformation in DH5α</p><br />
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<h1>July, 22nd</h1><br />
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<h2>Production Team</h2><br />
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<p><strong>Paola Gallardo.</strong> Site-directed mutagenesis (SDM): First try with <i>gltD**</i> and <i>fecI-fecR*</i>, using an overlapping PCR reaction. We analyzed the products of the first round. Fail. We'll try again</p><br />
<br />
<p><strong>David Caballero.</strong> Transformation of competent bacteria with ligation products we made the day before. We spread them in LB+Cm plates and let them grow overnight at 37ºC.</p><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>UPO1+2 was analized by 1.5% agarose gel electrophoresis again. Results showed three bands with the same size, so our candidate is negative. We started again with UPO1+2: digesting, binding, transforming.</p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/07_21Team:UPO-Sevilla/Notebook/07 212010-10-26T21:44:15Z<p>Dcabpra: </p>
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<h1>July, 21st</h1><br />
<br />
<h2>Production Team</h2><br />
<br />
<p><strong>Paola Gallardo.</strong> We isolated plasmid (minipreps.) from <i>fecR,</i> P<i>fecA,</i> <i>fecI-fecR-</i>P<i>fecA</i> and <i>fecR-</i>P<i>fecA,</i> and we digested them by using Kit Gingo. We made an analytic elctrophoresis (0'8%) of the products. Negative results for PfecA, positive results for <i>fecR,</i> <i>fecI-fecR-</i>P<i>fecA</i> and <i>fecR-</i>P<i>fecA.</i></p><br />
<br />
<p><strong>David Caballero.</strong> We got only one colony in each plate of P<i>fecA</i> and <i>gltB.</i> Colony analytic PCR reactions and 0.8% agarose gel electrophoresis analysis showed these colonies did not include mentioned parts. We deduced ligation had not been made in perfect conditions; surely the room temperature was too high. We prepared new ligation reactions for every part again and kept them at 16ºC overnight.</p><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>We have digested plasmid from three different colonies to check what parts we had. We likely have UPO1+UPO2 but we wanted to be completely sure so we will do again a digestion tomorrow. Moreover, we have tried to build UPO1+19 again so we have been ligating and transforming this afternoon.</p><br />
<br />
<h2>DryLab Team</h2><br />
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<p><strong>Wiki:</strong> Sponsors' footer included.</p><br />
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<h1>July, 20th</h1><br />
<br />
<h2>Production Team</h2><br />
<br />
<p><strong>Paola Gallardo.</strong> Purification of plasmids from inocula with <i>gltD**</i> and <i>fecI-fecR*</i> (we accidentally destroyed <i>fecI-fecR*-</i>P<i>fecA</i> and <i>fecR*-</i>P<i>fecA</i> inocula), digestion of these plasmids with restriction enzymes EcoRI and PstI. We ran and electrophoresis with the digestion products and we checked if we have isolated colonies that contain these plasmids. Setting up new inocula of <i>E. coli</i> with <i>fecR*</i>, P<i>fecA</i>, <i>fecI-fecR*-</i>P<i>fecA</i> y <i>fecR*-</i>P<i>fecA</i>; and finally, and isolation of the same bacterias in LB+Cm plates. We set up inocula of bacterias with <i>fecR</i>, P<i>fecA</i>, <i>fecI-fecR-</i>P<i>fecA</i> and <i>fecR-</i>P<i>fecA.</i></p><br />
<br />
<p><strong>David Caballero.</strong> Analysis of previous PCR reactions in 0.8% agarose gel electrophoresis. We had positives results for each amplified part: <i>fecA*</i> and <i>gltB.</i> The new parts were purified, digested and ligated in vectors with Cm resistance. Next we transformed competent bacteria with these parts and other ones: <i>fecI, </i>P<i>fecA,</i> <i>fecI-fecR*</i>, <i>fecA*</i> and <i>gltB.</i> We cultivated them in LB+Cm plates overnight to 37º.</p><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>Digestion of UPO16+3 to check if they had the plasmid. Now we have another new biobrick (UPO16+3)!</p><br />
<br />
<p>Transformation plates of UPO1+19 and UPO2+13 were quite weird... Religation plate had about 100 colonies (we expected not to have anything). Ligation of UPO 2+13 had 1 colony and UPO1+19 had anything. We did colony PCR again to check that we marked well every plate. The gel said that we weren’t wrong and that maybe we have a colony with the right plasmid of UPO2+13. We put inocula of UPO2+13 and spread in a plate. Maybe ligation are giving problems because of the heat in the lab.</p><br />
<br />
<h2>DryLab Team</h2><br />
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<p><strong>Wiki:</strong> Header created, sections added. </p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Notebook/07_20Team:UPO-Sevilla/Notebook/07 202010-10-26T21:25:13Z<p>Dcabpra: </p>
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<h1>July, 20th</h1><br />
<br />
<h2>Production Team</h2><br />
<br />
<p><strong>Paola Gallardo.</strong> Purification of plasmids from inocula with <i>gltD**</i> and <i>fecI-fecR*</i> (we accidentally destroyed <i>fecI-fecR*-</i>P<i>fecA</i> and <i>fecR*-</i>P<i>fecA</i> inocula), digestion of these plasmids with restriction enzymes EcoRI and PstI. We ran and electrophoresis with the digestion products and we checked if we have isolated colonies that contain these plasmids. Setting up new inocula of <i>E. coli</i> with <i>fecR*</i>, P<i>fecA</i>, <i>fecI-fecR*-</i>P<i>fecA</i> y <i>fecR*-</i>P<i>fecA</i>; and finally, and isolation of the same bacterias in LB+Cm plates. We set up inocula of bacterias with <i>fecR</i>, P<i>fecA</i>, <i>fecI-fecR-</i>P<i>fecA</i> and <i>fecR-</i>P<i>fecA.</i></p><br />
<br />
<p><strong>David Caballero.</strong> Analysis of previous PCR reactions in 0.8% agarose gel electrophoresis. We had positives results for each amplified part: <i>fecA*</i> and <i>gltB.</i> The new parts were purified, digested and ligated in vectors with Cm resistance. Next we transformed competent bacteria with these parts and other ones: <i>fecI, </i>P<i>fecA,</i> <i>fecI-fecR*</i>, <i>fecA*</i> and <i>gltB.</i> We cultivated them in LB+Cm plates overnight to 37º.</p><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>Digestion of UPO16+3 to check if they had the plasmid. Now we have another new biobrick (UPO16+3)!</p><br />
<br />
<p>Transformation plates of UPO1+19 and UPO2+13 were quite weird... Religation plate had about 100 colonies (we expected not to have anything). Ligation of UPO 2+3 had 1 colony and UPO1+19 had anything. We did colony PCR again to check that we marked well every plate. The gel said that we weren’t wrong and that maybe we have a colony with the right plasmid of UPO2+13. We put inocula of UPO2+13 and spread in a plate. Maybe ligation are giving problems because of the heat of the lab.</p><br />
<br />
<h2>DryLab Team</h2><br />
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<p><strong>Wiki:</strong> Header created, sections added. </p><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Modeling/SignalingTeam:UPO-Sevilla/Modeling/Signaling2010-10-26T21:16:42Z<p>Dcabpra: </p>
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<h1>The signaling circuit</h1><br />
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<p><br />
The signaling circuit 3 described in the <a href="https://2010.igem.org/Team:UPO-Sevilla/Biobricks/Circuits">Circuit Section</a> has been modeled using Matlab Simbiology desktop. The following diagram shows the different parts of the model we have simulated:<br />
</p><br />
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<a href="https://2010.igem.org/Image:UPOModelv2.png"><br />
<img src="https://static.igem.org/mediawiki/2010/5/5b/UPOModelv2.png" width="700" alt="Simbiology model"/><br />
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<p><br />
For the level of detail considered, the main parts simulated are the following (the number correspond to the equations listed in the table in the next section):<br />
</p><br />
<br />
<ol><br />
<li> Generation of L_aspartate induced by AAL</li><br />
<li> Diffusion of L_aspartate through the cell wall</li><br />
<li> Transcription of the aspA, promoted by FecI_a (active)</li><br />
<li> Translation of aspA</li><br />
<li> Activation of FecI, induced by the activation of FecR</li><br />
<li> Activation of FecR induced by FecA-PrhA </li><br />
<li> Plant cell wall lingand, FecA-PrhA binding</li><br />
</ol><br />
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<h1>Reactions</h1><br />
<br />
<p><br />
The reaction equations for the previous parts, and the reactions rates associated, are summarized in the following table:<br />
</p><br />
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<table xmlns="" id="modelContentsReactionTable" width="100%" border="5" cellpadding="5" cellspacing="0" class="dataTable"><tbody><br />
<tr><th width="5%">#</th><th>Reaction</th><th>ReactionRate</th><th>Active</th></tr><br />
<tr class="evenRow"><td>1</td><td>ecoli.ammonia + ecoli.fumarate + ecoli.AAL &lt;-&gt; ecoli.L_aspartate + ecoli.AAL</td><td>k1*ecoli.ammonia*ecoli.fumarate*ecoli.AAL - k2*ecoli.L_aspartate*ecoli.AAL</td><td>true</td></tr><br />
<br />
<tr class="oddRow"><td>2</td><td>ecoli.L_aspartate &lt;-&gt; medium.L_aspartate</td><td>kWallDiffusion*ecoli.L_aspartate - kWallDiffusionBack*medium.L_aspartate</td><td>true</td></tr><br />
<br />
<tr class="evenRow"><td>3</td><td>ecoli.DNAaspA + ecoli.FecI_a -&gt; ecoli.ARNm_aspA + ecoli.DNAaspA + ecoli.FecI_a</td><td>kTranscript*ecoli.DNAaspA*ecoli.FecI_a</td><td>true</td></tr><br />
<br />
<tr class="oddRow"><td>4</td><td>ecoli.ARNm_aspA -&gt; ecoli.AAL + ecoli.ARNm_aspA</td><td>kTranslation*ecoli.ARNm_aspA</td><td>true</td></tr><br />
<br />
<tr class="evenRow"><td>5</td><td>ecoli.FecR_a + ecoli.FecI &lt;-&gt; ecoli.FecI_a + ecoli.FecR_a</td><td>kFecIActivation*ecoli.FecR_a*ecoli.FecI - kFecIDeactivation*ecoli.FecI_a*ecoli.FecR_a</td><td>true</td></tr><br />
<br />
<tr class="oddRow"><td>6</td><td>ecoli.FecR + ecoli.[ligand:FecA-PrhA] &lt;-&gt; ecoli.FecR_a + ecoli.[ligand:FecA-PrhA]</td><td>kFecRActivation*ecoli.FecR*ecoli.[ligand:FecA-PrhA] - kFecRDeactivation*ecoli.FecR_a*ecoli.[ligand:FecA-PrhA]</td><td>true</td></tr><br />
<br />
<tr class="evenRow"><td>7</td><td>plant_cell_wall.ligand + ecoli.[FecA-PrhA] &lt;-&gt; ecoli.[ligand:FecA-PrhA]</td><td>kCellBinding*plant_cell_wall.ligand*ecoli.[FecA-PrhA] - kCellUnbinding*ecoli.[ligand:FecA-PrhA]</td><td>true</td></tr><br />
</tbody><br />
</table><br />
<br />
<h1>Simulations</h1><br />
<br />
<p>The following figure shows the typical evolution of the output of the system (the generated chemoattractant) againts the inputs (the wall cells ligand and the FecA-PrhA components on the outer membrane)</p><br />
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<br />
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<center><br />
<a href="https://2010.igem.org/Image:UPOSimulation.png"><br />
<img src="https://static.igem.org/mediawiki/2010/9/9f/UPOSimulation.png" width="500" alt="Simbiology model"/><br />
</a><br />
</center><br />
<br />
<h1>Analysis</h1><br />
<br />
<h2>Sensibility</h2><br />
<br />
<p><br />
Simbiology allows to compute the sensibility of the system against the different parameters. <br />
</p><br />
<br />
<p><br />
The following figure shows the sensibility of all state variables (molecules of the different species considered) with respect to all the parameters.<br />
</p><br />
<br />
<center><br />
<a href="https://2010.igem.org/Image:UPOSensibilityAll.png"><br />
<img src="https://static.igem.org/mediawiki/2010/2/21/UPOSensibilityAll.png" width="500" alt="Simbiology model"/><br />
</a><br />
</center><br />
<br />
<p><br />
What the analysis reveal is that the system is quite insensitive to changes in the parameters. This is due mainly to the nature of the transduction signals. The promoters act as a kind of "switch". This means that, provided these promoters reach certain levels, the other parts of the circuits are activated, even if the levels are not equal. <br />
</p><br />
<br />
<p><br />
This analysis is referred to the steady-state of the system. Some parameters do not affect the final steady-state number of molecules, but on the other hand affects the velocity of the system in the transition. This can be seen in the following paragraphs.<br />
</p><br />
<br />
<h2>Scanning of Parameters</h2><br />
<br />
<p><br />
If we perform a scan over several values of some parameters it can be seen the influence of these parameters on the output. For instance, in the following figure it can be seen how the parameter <i>kCellBinding</i> (the "force" of the binding with the cell wall) affects the final output of the system (<i>medium.L_aspartate</i>, the amount of chemoattractant), for several scales of magnitude.<br />
</p><br />
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<center><br />
<a href="https://2010.igem.org/Image:UPOScanBinding.png"><br />
<img src="https://static.igem.org/mediawiki/2010/8/84/UPOScanBinding.png" width="700" alt="Simbiology model"/><br />
</a><br />
</center><br />
<br />
<p><br />
This parameter affects the velocity of the system, but not in great deal. In the following, the same analysis is done for the four main steps in the generation of the chemoattractant (for quite different orders of magnitude):<br />
</p><br />
<br />
<ol><br />
<li> Transcription of the aspA, promoted by FecI_a (active)</li><br />
<li> Translation of aspA</li><br />
<li> Activation of FecI, induced by the activation of FecR</li><br />
<li> Activation of FecR induced by FecA-PrhA </li><br />
</ol><br />
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<a href="https://2010.igem.org/Image:UPOScanBindFecIFecRTranscriptTranslation.png"><br />
<img src="https://static.igem.org/mediawiki/2010/1/19/UPOScanBindFecIFecRTranscriptTranslation.png" width="700" alt="Simbiology model"/><br />
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</div></div>Dcabprahttp://2010.igem.org/Team:UPO-Sevilla/Project/ResultsTeam:UPO-Sevilla/Project/Results2010-10-26T21:11:54Z<p>Dcabpra: </p>
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<h1>Results</h1><br />
<br />
<h2>Building devices</h2><br />
<br />
<p>During this summer we tried to build a lot of devices (17 exactly) but we finally realized that they were too many for our first time. We had many problems assembling parts and checking the constructions: colony PCR did not work (or it just worked sometimes), digestion enzymes did not cut always, we could hardly obtain new parts by PCR or site-directed mutagenesis products, etc. We think most problems were caused because of the lab conditions. We worked in a practices lab and the material we used was not the best. When we noticed that the Jamboree date was too close, we had to focus in some devices and forget assembling others. <strong>Prioritizing</strong> some devices helped us to finish proposed devices. Our work moved forward faster.</p><br />
<br />
<p>You can see below the devices we finally built and what they are for.</p><br />
<br />
<ul><br />
<li><strong>Device 6</strong>. Quantify induction of P<i>fecA</i> promoter by GFP fluorescence.</li><br />
<br />
<img class="centerMedium" src="https://static.igem.org/mediawiki/2010/6/6a/BacterialCrowdingMiniDevice6.png" alt="Device 6 of Bacterial Crowding project"/><br />
<br />
<li><strong>Device 8</strong>. Production of chemoattractant aspartate mediated by signal transduction Circuit 2 in response to ferric citrate (because we could not assemble Circuit 3).</li><br />
<br />
<img class="centerMedium" src="https://static.igem.org/mediawiki/2010/c/c2/BacterialCrowdingMiniDevice8.png" alt="Device 8 of Bacterial Crowding project"/><br />
<br />
<li><strong>Device 9</strong>. Constitutive production of chemoattractant aspartate.</li><br />
<br />
<img class="centerMedium" src="https://static.igem.org/mediawiki/2010/5/58/BacterialCrowdingMiniDevice9.png" alt="Device 9 of Bacterial Crowding project"/><br />
<br />
<li><strong>Device 14</strong>. Production of chemoattractant salicylate (for <i>P. putida</i>) mediated by signal transduction circuit 2 in response to ferric citrate (because we could not assemble Circuit 3).</li><br />
</ul><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/8/86/BacterialCrowdingMiniDevice14.png" alt="Device 14 of Bacterial Crowding project"/><br />
<br />
<p>As you can see, we could not assemble any device related to the <a href="https://2010.igem.org/Team:UPO-Sevilla/Project/Sensing" target="_blank">Sensing</a> part of the project. Until the last day we tried to assemble the device that codes FecA/PrhA outer membrane hybrid protein (Device 17) but we did not achieve it. It seemed that this protein could have some harmful effects in bacteria. We tried to express FecA/PrhA under the control of a middle-strong constitutive promoter. First we did that using a high copy vector, but we soon changed it to a low copy plasmid. Nevertheless we never obtained colonies that harvest the hybrid protein and could survive. Two days before Wiki freezing we looked up any inducible expression vector we could use instead of our constitutive promoter. We found <a href="https://2010.igem.org/Team:UPO-Sevilla/Project/Sensing" target="_blank">TetR repressed POP/RIPS generator</a>, which showed suitable features, but we did not have enough time to test it before the Jamboree date. Anyhow, we thought that a good way to solve outer membrane protein expression problems is to use inducible promoters.</p><br />
<br />
<img class="centerMedium" src="https://static.igem.org/mediawiki/2010/c/c5/BacterialCrowdingMiniDevice17.png" alt="Device 17 of Bacterial Crowding project"/><br />
<br />
<h2>Chemotaxis assays</h2><br />
<br />
<p>The main way to test our project was to use chemotaxis assays. Over August we started to read as much articles about chemotaxis assays as we found. We soaked of these processes and tested some of them. In <a href="https://2010.igem.org/Team:UPO-Sevilla/Project/Sensing" target="_blank">Assay</a> site you can read more about all the different kinds of experiments we performed. Also we modified some of this assays.</p><br />
<br />
<p>When we started we used three bacterial strains: <i>E. coli</i> K-12, <i>Pseudomonas sp</i>. and <i>Pseudomonas putida</i> G7; and three chemoattractans, glutamate, aspartate and salycilate. Little by little we narrowed the possibilities to <i>E. coli</i> K-12 as strain and <strong>aspartate</strong> as attractant, because of its high expected chemotactic response. However, we could not achieve good results in our chemotaxis assays using tip boxes as chemotaxis chambers and needles instead of capillaries. So we changed media conditions, needle thickness, we did different dilutions… but successful results did not arrive. Finally we realized the chance involved in our misfortune, our <i>E. coli</i> strain got a mutation which did not allow it to move or sense chemotaxis stimulus. We checked it by using a soft agarose plate assay.</p><br />
<br />
<p>Then we changed our strain for other known motile <i>E. coli</i> RP437 strain which came from <a href="http://chemotaxis.biology.utah.edu/Parkinson_Lab/people/people.html" target="_blank">Sandy Parkinson lab</a> (chemotaxis researcher). Using the new strain we performed <strong>lower scale assays</strong> (1&#956;l capillaries, no more that 1ml of bacterial suspensions), optimized some conditions and we achieved our goal. By microscopy and dilution and spread the chemotactic response of the <i>E. coli</i> towards aspartate was characterized. </p><br />
<br />
<p>Here some pictures of a capillary assay of <i>E. coli</i> chemotaxis toward aspartate are shown. As you can see while time passes more bacteria accumulate inside the capillary and arround it. This fact clearly supports the chemotaxis response of <i>E. coli</i>.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/6/6f/BacterialCrowdingCapillaryAssayMicroscope.png" alt="Capillary assay Pictures"/><br />
<p class="caption"><i> <strong> Results of a capillary assay using microscope techniques</strong>. We can see that the chemotatic response toward aspartate is increasing as time passes by. Also there are major differences between the control without aspartate and the control with aspartate.</i></p><br />
<br />
<p>In the last chemotaxis assay we performed before Wiki freezing we used as chemotaxis chamber a <strong>flow-chamber</strong> with three channels and <strong>1&#956;l glass capillaries</strong>. In both edges of each channel there was a capillary, one with aspartate and other filled with buffer. We carried out experiments with two chambers, one was incubated at room temperature during 1h and the other at 30ºC. After incubation capillaries were cleaned with water and their contents were diluted and spread in agar plates. Then plates were incubated overnight at 37ºC and counted. Below results of this assay are shown. The response to aspartate is much higher than to buffer and also the standard deviation is lower when the temperature is fixed at 30ºC.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/a/a1/BacterialCrowdingChemotaxisAssayResults.png" alt="Bacterial Crowding Chemotaxis Assay Results"/><br />
<br />
<h2>Producing bacteria</h2><br />
<br />
<p> The Bacterial Crowding project included sensing non-diffusible signals and <strong>producing</strong> a <strong>chemoattractant</strong> as response. In spite of the fact that we could not work with the sensing part of the project we wanted to test if our modified bacteria were able to produce enough chemoattractant to induce chemotactic behaviour in their partners. Although we had two different built chemoattractant production circuits, for salycilate and aspartate, we focused on the generation of aspartate to attract <i>E. coli</i>, due to its higher chemotactic response. Anyway, we started to do assays with supernatant quite late and results were not available to upload to the Wiki page before Jamboree date.</p><br />
<br />
<p>We designed several growing media in which the expression of the plasmids could be analyzed, mainly media to induce P<i>fecA</i> promoter. We set up inocula in these media and when they were saturated, we centrifugated and reject the pellets. The supernatants would be used in chemotaxis assays.</p><br />
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<h1>Results</h1><br />
<br />
<h2>Building devices</h2><br />
<br />
<p>During this summer we tried to build a lot of devices (17 exactly) but we finally realized that they were too many for our first time. We had many problems assembling parts and checking the constructions: colony PCR did not work (or it just worked sometimes), digestion enzymes did not cut always, we could hardly obtain new parts by PCR or site-directed mutagenesis products, etc. We think most problems were caused because of the lab conditions. We worked in a practices lab and the material we used was not the best. When we noticed that the Jamboree date was too close, we had to focus in some devices and forget assembling others. <strong>Prioritizing</strong> some devices helped us to finish proposed devices. Our work moved forward faster.</p><br />
<br />
<p>You can see below the devices we finally built and what they are for.</p><br />
<br />
<ul><br />
<li><strong>Device 6</strong>. Quantify induction of P<i>fecA</i> promoter by GFP fluorescence.</li><br />
<br />
<img class="centerMedium" src="https://static.igem.org/mediawiki/2010/6/6a/BacterialCrowdingMiniDevice6.png" alt="Device 6 of Bacterial Crowding project"/><br />
<br />
<li><strong>Device 8</strong>. Production of chemoattractant aspartate mediated by signal transduction Circuit 2 in response to ferric citrate (because we could not assemble Circuit 3).</li><br />
<br />
<img class="centerMedium" src="https://static.igem.org/mediawiki/2010/c/c2/BacterialCrowdingMiniDevice8.png" alt="Device 8 of Bacterial Crowding project"/><br />
<br />
<li><strong>Device 9</strong>. Constitutive production of chemoattractant aspartate.</li><br />
<br />
<img class="centerMedium" src="https://static.igem.org/mediawiki/2010/5/58/BacterialCrowdingMiniDevice9.png" alt="Device 9 of Bacterial Crowding project"/><br />
<br />
<li><strong>Device 14</strong>. Production of chemoattractant salicylate (for <i>P. putida</i>) mediated by signal transduction circuit 2 in response to ferric citrate (because we could not assemble Circuit 3).</li><br />
</ul><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/8/86/BacterialCrowdingMiniDevice14.png" alt="Device 14 of Bacterial Crowding project"/><br />
<br />
<p>As you can see, we could not assemble any device related to the <a href="https://2010.igem.org/Team:UPO-Sevilla/Project/Sensing" target="_blank">Sensing</a> part of the project. Until the last day we tried to assemble the device that codes FecA/PrhA outer membrane hybrid protein (Device 17) but we did not achieve it. It seemed that this protein could have some harmful effects in bacteria. We tried to express FecA/PrhA under the control of a middle-strong constitutive promoter. First we did that using a high copy vector, but we soon changed it to a low copy plasmid. Nevertheless we never obtained colonies that harvest the hybrid protein and could survive. Two days before Wiki freezing we looked up any inducible expression vector we could use instead of our constitutive promoter. We found <a href="https://2010.igem.org/Team:UPO-Sevilla/Project/Sensing" target="_blank">TetR repressed POP/RIPS generator</a>, which showed suitable features, but we did not have enough time to test it before the Jamboree date. Anyhow, we thought that a good way to solve outer membrane protein expression problems is to use inducible promoters.</p><br />
<br />
<img class="centerMedium" src="https://static.igem.org/mediawiki/2010/c/c5/BacterialCrowdingMiniDevice17.png" alt="Device 17 of Bacterial Crowding project"/><br />
<br />
<h2>Chemotaxis assays</h2><br />
<br />
<p>The main way to test our project was to use chemotaxis assays. Over August we started to read as much articles about chemotaxis assays as we found. We soaked of these processes and tested some of them. In <a href="https://2010.igem.org/Team:UPO-Sevilla/Project/Sensing" target="_blank">Assay</a> site you can read more about all the different kinds of experiments we performed. Also we modified some of this assays.</p><br />
<br />
<p>When we started we used three bacterial strains: <i>E. coli</i> K-12, <i>Pseudomonas sp</i>. and <i>Pseudomonas putida</i> G7; and three chemoattractans, glutamate, aspartate and salycilate. Little by little we narrowed the possibilities to <i>E. coli</i> K-12 as strain and <strong>aspartate</strong> as attractant, because of its high expected chemotactic response. However, we could not achieve good results in our chemotaxis assays using tip boxes as chemotaxis chambers and needles instead of capillaries. So we changed media conditions, needle thickness, we did different dilutions… but successful results did not arrive. Finally we realized the chance involved in our misfortune, our <i>E. coli</i> strain got a mutation which did not allow it to move or sense chemotaxis stimulus. We checked it by using a soft agarose plate assay.</p><br />
<br />
<p>Then we changed our strain for other known motile <i>E. coli</i> RP437 strain which came from <a href="http://chemotaxis.biology.utah.edu/Parkinson_Lab/people/people.html" target="_blank">Sandy Parkinson lab</a> (chemotaxis researcher). Using the new strain we performed <strong>lower scale assays</strong> (1ul capillaries, no more that 1ml of bacterial suspensions), optimized some conditions and we achieved our goal. By microscopy and dilution and spread the chemotactic response of the <i>E. coli</i> towards aspartate was characterized. </p><br />
<br />
<p>Here some pictures of a capillary assay of <i>E. coli</i> chemotaxis toward aspartate are shown. As you can see while time passes more bacteria accumulate inside the capillary and arround it. This fact clearly supports the chemotaxis response of <i>E. coli</i>.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/6/6f/BacterialCrowdingCapillaryAssayMicroscope.png" alt="Capillary assay Pictures"/><br />
<p class="caption"><i> <strong> Results of a capillary assay using microscope techniques</strong>. We can see that the chemotatic response toward aspartate is increasing as time passes by. Also there are major differences between the control without aspartate and the control with aspartate.</i></p><br />
<br />
<p>In the last chemotaxis assay we performed before Wiki freezing we used as chemotaxis chamber a <strong>flow-chamber</strong> with three channels and <strong>1ul glass capillaries</strong>. In both edges of each channel there was a capillary, one with aspartate and other filled with buffer. We carried out experiments with two chambers, one was incubated at room temperature during 1h and the other at 30ºC. After incubation capillaries were cleaned with water and their contents were diluted and spread in agar plates. Then plates were incubated overnight at 37ºC and counted. Below results of this assay are shown. The response to aspartate is quite higher than to buffer and also the standard deviation is lower when the temperature is fixed at 30ºC.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/a/a1/BacterialCrowdingChemotaxisAssayResults.png" alt="Bacterial Crowding Chemotaxis Assay Results"/><br />
<br />
<h2>Producing bacteria</h2><br />
<br />
<p> The Bacterial Crowding project included sensing non-diffusible signals and <strong>producing</strong> a <strong>chemoattractant</strong> as response. In spite of the fact that we could not work with the sensing part of the project we wanted to test if our modified bacteria were able to produce enough chemoattractant to induce chemotactic behaviour in their partners. Although we had two different built chemoattractant production circuits, for salycilate and aspartate, we focused on the generation of aspartate to attract <i>E. coli</i>, due to its higher chemotactic response. Anyway, we started to do assays with supernatant quite late and results were not available to upload to the Wiki page before Jamboree date.</p><br />
<br />
<p>We designed several growing media in which the expression of the plasmids could be analyzed, mainly madia to induce P<i>fecA</i> promoter. We set up inocula in these media and when they were saturated, we centrifugated and reject the pellets. The supernatants would be used in chemotaxis assays.</p><br />
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<h1>July, 14th</h1><br />
<br />
<h2>Production Team</h2><br />
<br />
<p><strong>Paola Gallardo.</strong> We only had colonies in three plates: no-ligation control of the vector pBS1C3, plasmid with <i>gltD**</i> and plasmid with <i>fecI-fecR*-</i>P<i>fecA</i>. Results were not good at all. We ran an analytic electrophoresis of all the used parts to make sure that there were no mistakes in the ligation reaction, and the results were correct. A new ligation reaction was made with the same parts and vectors, and we waited to the next day.</p><br />
<br />
<p><strong>David Caballero.</strong> Repetition of electrophoresis in the same conditions, but time, 45’. Results were more favorable in this occasion. There were five spots: <i>fecI, gltD**, fecI-fecR*, fecI-fecR*-PfecA</i> and <i>fecR*-PfecA</i>. Due to their thickness we decided to purify only DNA from PCR reactions for parts <i>fecI, fecI-fecR*-PfecA</i> and <i>fecR*-PfecA</i>. It was made using GFX kit.</p><br />
<br />
<h2>Assembly Team</h2><br />
<br />
<p>Finally we saw the results today. There wasn’t any colony on any LB+Cm plate so far. The chances are that there was some problem with the antibiotic; we will try to venture which mistakes were made.</p><br />
<br />
<ol><br />
<li> Electrophoresis with the purified DNA. So far, results show that all the digestion was correctly made, except for UPO 16, so in that case we will have to repeat the procedure tomorrow.</li><br />
<br />
<li>Biobricks Ligation: in the same way we did the day before. They shall be resting all the night long and the next day we would transform them</li><br />
</ol><br />
<br />
<h2>DryLab Team</h2><br />
<br />
<p><strong>Web:</strong> Adding diverse sections and banners</p><br />
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<h1>Introduction</h1><br />
<br />
<br />
<p>Bacteria can sense a lot of different stimuli. They have to detect changes in their environment and interpret them. Bacteria use different receptors to sense ionic state, chemicals, pH, nutrients, lights… and, when a stimuli is caused, they use an intracellular signaling pathway to produce an specific response. Usually, signals that bacteria can sense are diffusible-signals. However, we will use the first known system that senses a non-diffusible signal: the <strong>Prh system</strong> of <i>Ralstonia solanacearum</i>. This system detects an unknown polysaccharide ligand of plant cell walls and activates a signal transduction cascade that, in original system, causes virulence gene expression. </p><br />
<br />
<p>The aim of the <strong>sensing circuits</strong> is to ensure that our system is able to detect specifically the plant cells walls and causes the production of a chemoattractant through signal transduction pathways and its diffusion through cell membranes. In order to achieve effective bacterial accumulation around plant cells, we have designed four different circuits that attempt to prevent possible different problems. We have combined different regulated proteins from <i>Escherichia coli</i> and <i>Ralstonia solanacearum</i> with specific biobricks, and we have replaced virulence genes by chemoattranctant production genes. So, despite of <i>R. solanacearum</i> phito-patogenicity, the fact of working with biobricks avoids us to have to work with the security measures required by this spaecie. </p><br />
<br />
<p>A future improvement for our project would be to use an <strong>adhesin</strong> to keep the bacteria attached to the plant cell wall, which actually happend in <i>R.solanacearum</i>. We expect our system works without it.</p><br />
<br />
<br />
<br />
<h3>Brief description of <i>Ralstonia solanacearum</i>:</h3><br />
<br />
<p><i><strong>Ralstonia solanacearum</strong></i> belongs to the family of Gram-negative phyto-pathogens. This bacterium causes great losses in crops worldwide in tropical, subtropical and temperate environments. The genes involved in virulence are known as <i>hrp</i> (hypersensitive response and pathogenicity) and they are induced by contact with various plant species from three different families of dicotyledonous (Solanaceae, Cruciferae and legumes), including both host and non host species. These genes encode a type III secretion system (TTSS), required to develop the disease in the host or the hypersensitive response (HR) in resistant and non-host plants. The HR is a defense mechanism that certain plant species possess, in which cells infected by a pathogen are killed to prevent spread to healthy tissue. </p><br />
<br />
<p>The secretion system is expressed only when there is a <strong>physical interaction</strong> between <i>R. solanacearum</i> and the plant cell. PrhA (plant regulator of hrp genes), the protein that recognizes the plant cell ligand, is an outer membrane receptor that shows homology with some TonB-dependent siderophore receptors. However, PrhA is not involved in the bacteria-plant cell adhesion, but only in sensing. The interaction of bacteria with plant cells occurs in two steps: first, <i>R. solanacearum</i> binds to the cell surface. This union is independent of PrhA or any protein encoded by <i>hrp</i>. Once the union has been established, the PrhA receiver can detect an accessible ligand, which increases the transcription of the <i>hrp</i> regulatory gene. Induction of <i>hrp</i> expression is very fast, around 90 minutes, a period much shorter than generation time of <i>R. solanacearum</i> in optimal conditions. </p><br />
<br />
<p>PrhA fundamental feature is that it is the first known bacterial receptor that can detect a <strong>non-diffusible signal</strong> present in plant cell walls. The possibility of attaching bacteria to a specific tissue was what made us choose the Ralstonia system for our project. The induction of the expression of virulence hrp regulon integrates a complex signaling cascade that begins in the PrhA outer membrane protein. PrhA transduces the contact-dependent signal through a complex regulatory cascade composed of PrhR, PrhI, PrhJ, HrpG and HrpB. Finally, HrpB activates the expression of <i>hrp</i>, comprising the TTSS structural genes and genes that encode effector proteins that travel through the TTSS.</p><br />
<br />
<br />
<br />
<h1>Signal Transduction Circuits</h1><br />
<br />
<h2>Prh system of <i>Ralstonia solanacearum</i></h2><br />
<br />
<h3>Brief description of the original Prh system:</h3><br />
<br />
<p>The <strong>Prh system</strong> integrates the genes involved in the control of expression of <strong><i>hrp</i> virulence genes</strong> of <i>Ralstonia solanacearum</i>. <i>hrp</i> gene encodes a type III secretion system, necessary to develop disease in their hosts. The induction of these genes integrates a complex signaling network that begins when the bacteria and the plant cell contact. This signaling mechanism is composed of PrhA, PrhR and PrhI proteins and other regulators that, as a last resort, activate the expression of <i>hrp</i> or <i>hrc</i> (conserved hrp genes) genes.</p><br />
<br />
<p>The induction of virulence genes occurs when PrhA contacts with a plant cell. PrhA is an outer membrane protein that recognizes an unknown non-diffusible signal from the plant cell wall. PrhA-ligand binding causes that the periplasmic exposed N-terminal end of PrhA interacts with the carboxy terminal end of PrhR (an inner membrane protein) in the periplasm, transmitting the signal across the cytoplasmic membrane. In the cytoplasm inactive PrhI is actived by PrhR interaction by a still unkown mechanism.</p><br />
<br />
<p>The <i>prhIR</i> gene expression is induced in coculture with plant cells due to unknown environmental signal PrhA independent. PrhI is an ECF (extracytoplasmic function) sigma factor that, when it is activated, binds to RNA polymerase core enzyme and directs the polimerase to the promoter region of <i>PrhJ</i> gene to initiate transcription. In <i>R. solanacearum</i>, PrhJ protein induces <i>hrpG</i> transcription, which activates expression of <i>hrpB</i> gene and finally expresses <i>hrp</i> and <i>hrc</i> virulence genes.</p><br />
<br />
<p>The <strong>PrhA-PrhR-PrhI</strong> module of <i>Ralstonia</i> works similarly to FecA-FecR-FecI module of <i>E.coli</i>, with both similar sequences. PrhA shows homology with several members of the family of siderophore outer membrane receptors (as is the case of FecA). Two of the three boxes that this family of proteins presents (TonB-box, box II and boxIII) are well conserved and correctly located in PrhA. PrhR has a transmembrane domain (TM) in the same position as FecR and both proteins have a similar orientation. In addition, two of the three tryptophan residues of the N-terminal end of FecR required to activate FecI are present in PrhR. However, unlike most of the siderophores, both <i>prhIR</i> and <i>prhA</i> lack Fur-boxes which are necessary for the regulation in function of the internal iron status.</p><br />
<br />
<p>Another striking difference between PrhAIR and FecAIR is their gene organization: while there is a physical grouping between genes of FecAIR, in PrhAIR system, <i>prhA</i> constitutes a monocistronic operon at the left edge of <i>hrp</i> gene cluster and <i>prhIR</i> is on the right side of cluster, both <i>prhA</i> and <i>prhIR</i> separated by virulence genes. Moreover, in contrast to the Fec system where FecA is activated by FecI and repressed by Fur, PrhA is always expressed at very low level in the presence of the inducing signal and is PrhI independent.</p><br />
<br />
<br />
<br />
<h3>Circuit 1:</h3><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/4/4e/BacterialCrowdingCircuit1.png" alt="Bacterial Crowding Circuit 1" /><br />
</div><br />
<br />
<p>In circuit 1 we wanted to use Prh system to transduce plant cell wall signals in chemoattractant synthesis. This first circuit integrates regulatory components PrhA-PrhI-PrhR and PprhJ of <i>R. solanacearum,</i> but they are transfered to <i>E. coli.</i> Genes required for synthesis and excretion of the chemoattractant are under PprhJ promoter control.</p><br />
<br />
<p>Because the Prh system is not fully characterized, unknown elements could be involved in, preventing the correct transmittion of signals to P<i>prhJ</i>. Also, it is possible that it could not perform its usual answers when expressed in <i>E.coli</i>. For example, it could have problems setting PrhA protein in the outer membrane. For those reasons, we have designed other circuits wich use <i>E. coli</i> proteins in the signal cascade.</p> <br />
<br />
<div class="clear"></div><br />
<br />
<h2>Fec system of <i>Escherichia coli</i></h2><br />
<br />
<h3>Brief description of the original Fec system:</h3><br />
<br />
<p><strong>Fec system</strong> includes genes involved in regulation and expression of <i>E. coli</i> iron transporters. <i>fecABCDE</i> genes express the <strong>ferric citrate transporter</strong> when bacteria iron status is low or deficient. The induction of genes <i>fecABCDE</i> integrates a signaling cascade that begins at the cell surface and is extended to the cytoplasm. To do this, three specific proteins are involved : FecA in outer membrane, FecR in cytoplasmic membrane and FecI in cytoplasm. This module <strong>FecA-FecR-FecI</strong> is known as a signal transduction system between three compartments (outside, periplasm and cytoplasm).</p><br />
<br />
<p>The signaling pathway begins when the outer membrane receptor FecA binds to its ligand, ferric dicitrate. This binding causes structural changes in FecA that allow the interaction of its amino terminal end to the carboxy terminal end of FecR in the periplasm. FecR, a transmembrane protein, transmits the signal to the cytoplasm, where it activates FecI. FecI is an extracitoplasmatic function (ECF) sigma factor that, when activated, binds to core RNA polymerase and directs the complex to the upstream promoter of <i>fecABCDE</i> transport genes to initiate transcription. </p><br />
<br />
<p>In addition, the transcription of regulatory genes <i>fecIR</i> is controlled by the internal iron status through the Fur repressor. When the Fur protein is loaded with Fe 2+, it represses <i>fecIR</i> transcription and prevents the <i>fec</i> gene expression. Therefore, the <i>fec</i> transport gene transcription is subjected to a double control: first, cells detect iron deficiency. Then, regulatory proteins FecI and FecR are synthesized, which, if ferric citrate is available, initiate the transcription of <i>fec</i> transport genes.</p><br />
<br />
<p>Dicitrate ferric transport through the outer membrane requires an energy transduction complex consisting of TonB, ExbB and Exb cytoplasmic membrane proteins.</p><br />
<br />
<h3>Circuit 2:</h3><br />
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<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/a/a0/BacterialCrowdingCircuit2.jpg" alt="Bacterial Crowding Circuit 2" /><br />
</div><br />
<br />
<p>In our second circuit, the iron transport genes (<i>fecABCDE</i>) are replaced by genes required for synthesis and excretion of chemoattractant. Those genes remain under control of the PfecABCDE promoter, being regulated by the FecA-FecI-FecR module, which depends on internal iron status and external ferric citrate concentration.</p><br />
<br />
<p>This circuit has the advantage that, besides being well characterized, is present in wild type <i>E. coli</i>. However, this system is not specifically directed to plant tissues and would be regulated by iron status of the bacteria and the environment. Nevertheless, the second signal transduction circuit could be use as a control of the chemoattractant synthesis. This way, if the plant cell wall signal is not properly transduced, we could induce the chemoattractant synthesis by changing medium conditions.</p><br />
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<div class="clear"></div><br />
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<h2>(FecA/PrhA)-FecI-FecR hybrid protein system</h2><br />
<br />
<h3>Circuit 3:</h3><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/9/9d/BacterialCrowdingCircuit3.jpg" alt="Bacterial Crowding Circuit 3" /><br />
</div><br />
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<p>Our third circuit uses the FecA and PrhA <strong>sequence homology</strong>. We have designed an <strong>hybrid protein</strong> in order to detect the plant cell ligands and transmit the signal to FecR. The hybrid protein contains most of PrhA and the N-terminal end of FecA; binding both proteins by a shared sequence near the Ton-box. The signal would be transmited through the interaction between the periplasmic exposed N-terminal extension of FecA and the C-terminal part of FecR.</p><br />
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<p>The third circuit would allow us to sense a non-diffusible signal and to transduce it using an <i>E. coli</i> system without problems of expression and function.</p><br />
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<p>We have focused in this circuit. Below you can see a detailed description of hybrid protein structure.</p><br />
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<div class="clear"></div><br />
<br />
<h2>PrhA-fecI-FecR hybrid system</h2><br />
<br />
<h3>Circuit 4:</h3><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/7/73/BacterialCrowdingCircuit4.png" alt="Bacterial Crowding Circuit 4" /><br />
</div><br />
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<p>Due to the proximity in the life tree, the similarities between N-terminal extension of PrhA and FecA is significant, in particular the amino acid sequence Gx10(L,A)L(D,Q,A)G(S,T)L is well conserved. Also PrhR shows sequence similarity with FecR (27% identity, 43% similarity). Cause this information we wanted to test if the interaction between these systems was possible without modification.</p><br />
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<p>However, this construction is largely a test and we had not enough time to permorm it.</p><br />
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<div class="clear"></div><br />
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<h1>Outer Membrane Protein Structures</h1><br />
<br />
<p>Now it is going to be shown the structure of the outer membrane proteins which starts the signal transduction in sensing systems described before. It is interesting to study the structure and domains of FecA and PrhA before seeing the hybrid protein, in which we have focused our project mainly.</p><br />
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<h2>Fe(3+) dicitrate transport protein FecA</h2><br />
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<img class="center" src="https://static.igem.org/mediawiki/2010/7/78/BacterialCrowndingFecA01.png" alt="Crystal structure of the Outer Membrane Transporter FecA"/><br />
<p class="caption"><b><a src="http://www.pdb.org/pdb/explore/explore.do?pdbId=1KMO" target="_blank">Crystal structure of the Outer Membrane Transporter FecA.</a></b></p><br />
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<p><a href="http://www.uniprot.org/uniprot/P13036" target="_blank">FecA</a> is the outer membrane receptor protein in the Fe(3+) dicitrate transport system of <i>Escherichia coli</i>. It binds and transports ferric citrate, and it is required to initiate transcription of the <i>fecABCDE</i> transport operon but not the regulatory fecIR genes. This is a well-known protein, compound of 773 amino acids, whose main domains are shown below:</p><br />
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<img class="centerBig" src="https://static.igem.org/mediawiki/2010/6/63/BacterialCrowdingFecADomains.png" alt="FecA Domains"/><br />
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<p>The yellow left domain represents a <strong>signal peptide</strong> which takes from 1st to 33rd codon. The cleavage site of the signal peptidase has been found between residues 33 and 34<sup><a href="#Reference_Sensing01">[1]</a></sup>. Its function is to drive FecA protein to the outer membrane of <i>E. coli</i>, where the protein works.</p><br />
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<div class="imgLeft"><br />
<img class="ileft" src="https://static.igem.org/mediawiki/2010/8/8c/BacterialCrowdingFecATonB.png" alt="the structure of the periplasmic signaling domain of FecA by nuclear magnetic resonance" /><br />
</div><br />
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<p>The green illustration represents <strong>Secretin and TonB N-terminus Short Domain</strong> which takes from 57th to 107th codon. This domain is found at the N-terminus of the Secretins of the bacterial type II/III secretory system as well as the TonB-dependent receptor proteins. These proteins are involved in TonB-dependent active uptake of selective substrates. Thus, FecA interacts with TonB, which couples the electrochemical potential of the cytoplasmic membrane to active transport of ferric citrate across the outer membrane. The TonB box undergoes a substrate-induced disorder transition which produces an aqueous exposed, highly disordered protein fragment, which probably regulates transporter–TonB interactions<sup><a href="#Reference_Sensing02">[2]</a></sup>.</p><br />
<br />
<p>It is usual to find the TonB domain nearby signal and Plug domains. It is a common domain organization. At the left it is shown the structure of the periplasmic signaling domain of FecA by nuclear magnetic resonance. </p><br />
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<p>Between both before domains it is a flexible 79-residue domain of FecA termed the <strong>NH2-terminal extension</strong>, which resides entirely within the periplasm. Its function is proposed to be to transmit the liganded status of the receptor to FecR<sup><a href="#Reference_Sensing03">[3]</a></sup>.</p><br />
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<div class="clear"></div><br />
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<p>In red color in the schematic representation it is shown the <strong>TonB-dependent Receptor Plug Domain</strong> which takes from 129th to 244th codon. The Plug domain has been shown to be an independently folding subunit of the TonB-dependent receptors. It acts as the channel gate, blocking the pore until the channel is bound by ligand. At this point it undergoes conformational changes that open the channel. Also ligand induces allosteric transitions which are propagated through the outer membrane by the plug domain, signaling the occupancy of the receptor in the periplasm. The plug domain is located inside a barrel, comprising five helixes, two &beta; strands, and a mixed four-stranded &beta; sheet. Also three loops of the Plug domain extend above the plane of the upper leaflet of the outer membrane<sup><a href="#Reference_Sensing03">[3]</a></sup>.</p><br />
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<img class="centerBig" src="https://static.igem.org/mediawiki/2010/0/04/BacterialCrowdingFecAUnligandedandFerric.png" alt="FecA Crystal structure"/><br />
<p class="caption"><b>a.</b>Crystal structure of ferric citrate transporter FecA in the unliganded form <br/><br />
<b>b.</b>Crystal structure of the outer membrane transporter FecA complexed with ferric citrate</p><br />
<br />
<p>Finally in the C-terminus there is a <strong>TonB Dependent Receptor Domain</strong> which takes from 525th to 773rd codon. The TonB dependent receptor domain is included in the 22-stranded &beta; barrel that traverse de outer membrane. The barrel of a TonB dependent receptor is a dynamic entity that actively participates in the energy-dependent siderophore uptake. This barrel has elipsoidal shape as you can see in before representations of FecA. Below it is shown the C-terminal domain of FecA, from 525th codon to the end.</p><br />
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<img class="centerSmall" src="https://static.igem.org/mediawiki/2010/9/9d/BacterialCrowdingFecAC.png" alt="C-terminal domain of FecA"/><br />
<p class="caption">C-Terminal domain of FecA (representation made with RasWin program)</p><br />
<br />
<p>The common domain organization represents TonB dependent receptor domain at the same time as Plug domain because the interaction between the receptor (FecA) and the ligand (dinuclear ferric citrate molecule) is performed by Plug domain and the barrel. Formation of the liganded complex carries out changes on the conformation of the barrel and the Plug domain of FecA.</p><br />
<br />
<h2>Outer membrane receptor protein PrhA</h2><br />
<br />
<p>PrhA is the only known protein able to detect a non-diffusible signal and transduce this information into the cell. It is compound of 770 amino acids and it was found not too long ago. This is why there is not many information about it. Not being well-known is a point to use the hybrid protein FecA/PrhA instead of it. Anyway, their main domains are shown in <a href="http://pfam.sanger.ac.uk/protein?acc=B7ZJG7" target="_blank">Pfam website</a>, but it is not possible to see its structure because it has not been modeled yet.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/d/dd/BacterialCrowdingPrhADomains.png" alt="PrhA Domains"/><br />
<br />
<p>Like in the case of FecA, PrhA has a putative <strong>signal peptide</strong> which takes from 1st to 35th codon. Its function would be direct PrhA to the outer membrane of <i>Rastonia solanacearum</i>. Despite its existence, you can not see it in the domain summary picture since it has not been well studied.</p><br />
<br />
<p>Next it is a not confirmed domain with unknown function which would take from the beginning of the protein to 130th amino acid. By now, it is called <strong>PfamB PB000342</strong> and its family was generated automatically from an alignment taken from Automatic Domain Decomposition Algorithm (<a href="http://ekhidna.biocenter.helsinki.fi/sqgraph/pairsdb/index_html" target="_blank">ADDA</a>). Since PrhA interacts with PrhR using its periplasmic domain, it is expected that this domain performs that function.</p><br />
<br />
<p>Then, PrhA presents the same domains that FecA: <strong>TonB-dependent Receptor Plug Domain</strong> (154 – 250 aa.) and <strong>TonB-dependent Receptor Domain</strong>(542 – 767 aa.), setting out the high similarities that exist between these two outer membrane proteins. Also their N-terminal extensions are quite similar as it was found by Marenda et al<sup><a href="#Reference_Sensing04">[4]</a></sup>.</p><br />
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<img class="centerBig" src="https://static.igem.org/mediawiki/2010/c/c8/BacterialCrowdingFerrecCitrateTree.png" alt="Ferric Citrate tree"/><br />
<p class="caption"> <strong> Phylogenetic tree of TonB-dependent receptors </strong>. The tree was constructed as described by Rakin et al. (1994). Circled numbers indicate the number of times (from the whole 100) a particular node was supported by bootstrap analysis. The proteins used in this analysis are referenced in Rakin et al. (1994).<sup><a href="#Reference_Sensing04">[4]</a></sup></p><br />
<br />
<p>PrhA has high similarities with TonB-dependent receptors, which needs to interact with the TonB protein to perform their functions. It shares two of the three main domains those proteins have. Nevertheless, PrhA is lacking of the periplasmic Secretin and TonB N-terminus Short Domain, the necessary domain to interact with TonB. In its place there is an unknown domain still not well studied. It will be required to continue studying this protein to know if TonB is necessary in its function and understand the evolution that TonB interaction domain suffered.</p><br />
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<h2>Hybrid protein FecA/PrhA</h2><br />
<br />
<p>Taking in advance that Prh system is not naturally expressed in <i>Escherichia coli</i> and that Prh system is not well-known, we decided to create a fusion protein. FecA/PrhA artificial coding sequence spans the first 92 codons of <i>fecA</i>, encoding the signal peptide, NH2 terminal extension and the proposed Ton-box, fused to the distal end of the <i>prhA</i> coding sequence at the conserved GSGL motif (aa. 89-92). We synthesized this biobrick using MrGene services, so we also optimized the sequence to be expressed in <i>Escherichia coli</i>.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/2/2c/BacterialCrowdingFecAPrhAHybrid.png" alt="PrhA, FecA and Hybrid Domains"/><br />
<br />
<p>Above is shown approximately the point where we fused FecA and PrhA proteins. To this way the hybrid protein include domains from both OM proteins:</p><br />
<br />
<ul><br />
<li><strong>Signal peptide of FecA</strong> which will help to the accurate emplacement of the hybrid protein in the outer membrane of <i>E. coli</i>.</li><br />
<br />
<li><strong>NH2-terminal extension of FecA</strong>. Including this domain of FecA means including the periplasmic signaling domain of this protein. The signal transfer between the OM and the IM proteins is performed between the N-terminus of FecA and the C-terminus of FecR (both are shown in the periplasmic). The hybrid protein includes the N-terminus of FecA so our expectation is that FecA/PrhA protein was able to interact with FecR.</li><br />
<br />
<li>Most of the <strong>Secretin and TonB N-terminus Short Domain of FecA</strong>. This domain helps FecA to interact with TonB. If TonB interaction is required for the OM-IM signal transfer, our hybrid protein includes this domain. Also, doing this fusion the hybrid protein loses the unknown function domain set in the N-terminus of PrhA. <br/><br />
<img class="centerSmall" src="https://static.igem.org/mediawiki/2010/2/21/BacterialCrowdingFecAN.png" alt="N-terminus short domain of FecA"/><br />
<p class="caption"><strong>N-terminus</strong> (aa. 34-92) <strong>of FecA.</strong> Here is shown the FecA contribution to the hybrid protein. Our aim is that this structure was able to interact with FecR without the rest of FecA protein. Representation made with RasWin.</p></li><br />
<br />
<li><strong>TonB-dependent Receptor Plug Domain of PrhA</strong>. In 89<sup>th</sup> codon there is a conserved motif which was used to fuse FecA with PrhA. The function of the Plug domain is to propagate allosteric transitions through the outer membrane signaling the occupancy of the receptor.</li><br />
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<li><strong>TonB Dependent Receptor Domain of PrhA</strong>. From the conserved motif GSGL to the end of the protein amino acids are the same that in PrhA protein. The hybrid protein includes most of the PrhA protein, from 92<sup>sd</sup> codon to the C-terminus. The aim of it is that FecA/PrhA was able to interact with the non-diffusible plant wall signal that PrhA detects. The mechanism of this interaction is unknown.</li><br />
</ul><br />
<br />
<p>As you can see we work with a lot of uncertainty cause of the unknown mechanisms that manage the process we work with. Anyhow, we hope that this hybrid protein allows to sense non-difusible signals (with PrhA domains) and to transduce it by the Fec pathway (using the N-terminus of FecA). If this happened we would not have any problem with other Prh protein because the signal would continue by FecR and FecI in the Fec pathway of <i>E. coli<i>.</p><br />
<br />
<h1>Bibliography</h1><br />
<br />
<ol><br />
<li id="Reference_Sensing01">Uwe Pressler, Horst Staudenmaier, Luitgard Zimmermann, And Volkmar Braun (1988), Genetics of the Iron Dicitrate Transport System of Escherichia coli. JOURNAL OF BACTERIOLOGY, June 1988, p. 2716-2724</li><br />
<br />
<li id="Reference_Sensing02">Miyeon Kim, Gail E. Fanucci, and David S. Cafiso (2007), Substrate-dependent transmembrane signaling in TonB-dependent transporters is not conserved. PNAS, July 17, 2007, vol. 104, no. 29, 11975–11980.</li><br />
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<li id="Reference_Sensing03">Andrew D. Ferguson, et al (2002). Structural Basis of Gating by the Outer Membrane Transporter FecA. Sience 295, 1715.</li> <br />
<br />
<li id="Reference_Sensing04">Marc Marenda, Belen Brito, Didier Callard, Stéphane Genin, Patrick Barberis, Christian Boucher and Matthieu Arlat (1998). PrhA controls a novel regulatory pathway required for the specific induction of Ralstonia solanacearum hrp genes in the presence of plant cells. Molecular Microbiology (1998) 27(2), 437–453.<br/><br />
<br />
</li> <br />
<br />
</ol><br />
<br />
<ul><br />
<li>Brito, B., Marenda, M., Barberis, P., Boucher, C., and Genin, S. 1999. <i>prhJ and hrpG: Two new components of the plant signal-dependent regulatory cascade controlled by PrhA in Ralstonia solanacearum</i>. Mol. Microbiol. 31:237-251.</li><br />
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<li>Marenda, M., Brito, B., Callard, D., Genin, S., Barberis, P., Boucher, C. A., and Arlat, M. 1998. <i>PrhA controls a novel regulatory pathway required for the specific induction of Ralstonia solanacearum hrp genes in the presence of plant cells</i>. Mol. Microbiol. 27:437-453.</li><br />
<br />
<li>Aldon, D., Brito, B., Boucher, C., and Genin, S. 2000. <i>A bacterial sensor of plant cell contact controls the transcriptional induction of Ralstonia solanacearum pathogenicity genes</i>. EMBO (Eur. Mol. Biol. Organ.) J. 19:2304-2314.</li><br />
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<li>Brito, B., Aldon, D., Barberis, P., Boucher, C., and Genin, S. 2002. <i>A Signal Transfer System Through Three Compartments Transduces the Plant Cell Contact-Dependent Signal Controlling Ralstonia solanacearum hrp Genes</i>. Molecular Plant-Microbe Interactions. Vol. 15, No. 2: 109/119</li><br />
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<li>Braun V, Mahren S, Sauter A. <i>Gene regulation by transmembrane signaling</i>. 2006. Biometals. 19(2):103-13</li><br />
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<li>Braun V, Mahren S, Ogierman M. 2003. <i>Regulation of the FecI-type ECF sigma factor by transmembrane signalling</i>. Curr Opin Microbiol. 6(2):173-80.</li><br />
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<li>Enz, S., Brand, H., Orellana, C., Mahren, S., and Braun, V. 2003. <i>Sites of Interaction between the FecA and FecR Signal Transduction Proteins of Ferric Citrate Transport in Escherichia coli</i> K-12. J. Bacteriol. Vol. 185, No: 133745–3752 </li><br />
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<li>Kim, M., Fanucci, G. E., and Cafiso, D. S. 2007. <i>Substrate-dependent transmembrane signaling in TonB-dependent transporters is not conserved</i>. PNAS. Vol. 104 N. 29: 11975/11980</li><br />
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<li>http://www.mikrobio.uni-tuebingen.de/ag_braun/research_areas.html</li><br />
<li>www.uniprot.org </li><br />
<li>http://pfam.sanger.ac.uk/ </li><br />
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</ul><br />
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