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- | ==Results==
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- | <html>
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- | <div class="liquid-slider" id="slider-id">
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- | <div>
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- | <h2 class="title"><img src="https://static.igem.org/mediawiki/2013/2/20/White_riding_icon.png" alt="" class="title-icon" />The Riding</h2>
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- | <h1 class="title2 riding-color"><img src="https://static.igem.org/mediawiki/2013/2/20/White_riding_icon.png" alt="" class="title2-icon riding-bg" />The Riding</h1>
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- |
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- | <p>
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- | In this synthetic symbiosis<b>, <i>C. elegans</i> </b>acts as a transport for engineered bacteria <b>(<i>Pseudomonas putida</i></b><i>) </i>in order to
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- | take them to the hotspot of interest, because bacteria are are not able to move fast in through solid or semi-solid substrates but they are very
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- | interesting from a biotechnological point of view, that is the reason why we though in this innovative mean of transport: the regulated <b> formation of a biofilm</b>.
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- | </p>
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- | <p>
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- | To achieve our goal, we constructed a BioBrick (see part: <u><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1112001">BBa_K1112001</a></u>)
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- | consisting in the coding sequence of the <b>hmsHFRS operon</b>, an adhesion operon natural from <i>Xenorhadbus nematophila</i> which allows the formation
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- | of a biofilm on the nematode <i>S. carpocapsae</i>, under the control of a nitrogen sensitive promoter (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K763003"><b>pGlnA, </b>characterized by the <b> 2012 Valencia Biocampus iGEM team</b></a>). (<b>Fig.1</b>)
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- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/1/1b/Vb_riding_1.png" style="width:700px;" alt="" />
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- | </p>
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- | <p>
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- | <h3>Controlling the mechanism</h3>
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- | Riding was a regulated process: with <b>low nitrogen </b>in the media, the promoter is activated and <i>hms</i> genes are expressed, triggering the <b>formation of the biofilm </b>over <i>C. elegans</i>; in contrast, with <b>high nitrogen </b>concentrations, such as the ones found in nutrient-rich hotspots, the promoter is repressed, so bacteria can <b>“get off</b>” the nematode <b>(Fig. 2)</b>.
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- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/2/20/Vb_riding_2.png" style="width:700px;" alt="" /><br/>
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- | The manufacturing of the plates can be seen in <a href="http://www.youtube.com/watch?v=5MIEVW6NedE">this video</a>.
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- | </p>
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- | <p>
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- | <h3>Biofilm formation in genetically-engineered bacteria</h3>
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- |
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- | Our original idea was to introduce the Biobrick in <b><i>Pseudomonas putida </i></b>, a bacterial species with wide applications in biotechnology. To do
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- | that, we cloned the construction (<b>Fig.1)</b> in the <b>pIZ1016 vector</b>, which has a replication origin compatible with <i>Pseudomonas</i>. We
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- | successfully performed the cloning <b>(Fig.3)</b>, but the efficiency of the transformation was too low, so haven’t been able to obtain <i>P. putida </i>
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- | transformants yet. This is probably a consequence of the length of the construction, 6,5 kb, which decreases transformation efficiency.
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- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/8/8d/Vb_riding_3.png" style="width:700px;" alt="" />
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- | </p>
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- | <p>
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- | But far from being disheartened, we decided to express the construction with <b><i>E. coli.</i> </b>We cloned the construction in the pUC57 vector,
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- | obtained transformant <i>E. coli</i>, and then grew them in medium with low nitrogen (0,6 g/L) in order to induce the formation of the biofilm. <i>C. elegans </i>was fed with these induced bacteria, and then several worms were isolated in order to check biofilm formation with <b>scanning electron microscopy (SEM) </b>imaging. As you can see in <b>Fig.4</b>, we actually did it! We observed a formation of an <i>E. coli </i>biofilm
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- | over the nematode!
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- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/0/03/Vb_riding_4.png" style="width:700px;" alt="" />
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- | </p>
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- | </div>
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- | <div>
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- | <h2 class="title"><img src="https://static.igem.org/mediawiki/2013/3/36/White_calling_icon.png" alt="" class="title-icon" />The Calling</h2>
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- | <h1 class="title2"><img src="https://static.igem.org/mediawiki/2013/3/36/White_calling_icon.png" alt="" class="title2-icon" />The Calling</h1>
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- | <p>
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- | <h3>Looking for an attractant</h3>
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- | When we were considering setting up a transport of bacteria was thought to be necessary to have a 'destination', a place to go. That destiny would be a <b>'hot spot' on a heterogeneous substrate</b> where <b><i>Caenorhabditis</i> <i>elegans</i></b> should lead right to that point the bacteria.
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- | <br/>
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- | Leveraging the powerful smell of the nematode, it was decided to try a number of attractants from various lists from web <a href="http://www.wormbook.org">www.wormbook.org</a> that could work as 'hot spot' of our experiment. Thus, using the<i> C. elegans</i> <b>chemotaxis, </b>we could direct transport.
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- | </p>
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- |
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- |
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- | <p>
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- | <h3>The attractants experiment</h3>
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- |
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- | The test would be carried creating our own plates on which half would be NGM unmodified and the other half part would be including soluble compounds before
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- | solidifying or after solidification in the case of volatiles. The list of modifications can be found in <b>Fig. 1</b>.
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- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/8/83/Calling_table1.png" style="width:700px;" alt="Table 1" />
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- | </p>
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- | <p>
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- | To place <b><i>C. elegans</i> </b>on the plate, different cuts were made on a fresh plate of NGM, the resulting small pieces were placed in the exact
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- | center of the 50% -50% plates to determine which side of the nematode preferred, one per Petri plate.
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- | </p>
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- | <p>
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- | The results after counting 2 replicates per attractant can be seen in <b>Fig. 2</b>.
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- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/c/ce/Calling_table2.png" style="width:700px;" alt="Table 2" />
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- | </p>
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- | <p>
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- | From <b>Table 2 </b>it could rule out many of the attractants that were thought viable.
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- | </p>
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- | <p>
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- | Volatile attractants were not a good choice to evaporate quickly (which is also limited to the field experiments).
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- | </p>
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- | <p>
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- | Another impediment arose. Once had already performed the experiments, it was decided that the promoter that will control the production of RNA interference
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- | in <i>Escherichia coli</i> would be controlled by nitrogen, amino acids had to be discard as attractants; it would modify controlled expression of E.coli.
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- | </p>
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- | <p>
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- | That left the MgSO<sub>4</sub> and hypoosmotic media as potential attractants.
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- | </p>
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- | <p>
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- | <h3>Attractant final choice</h3>
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- | Because the hyposmotic medium could interfere with the proper growth of bacteria (food of our nematode), the final decision was to choose the MgSO<sub>4</sub> as attractant.
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- | </p>
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- | <p>
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- | The question that arose at that time was: <i>How we can multiply the amount of MgSO<sub>4</sub> to increase efficiency?</i>
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- | </p>
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- | <p>
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- | <h3>MgSO<sub>4</sub> efficiency</h3>
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- |
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- | Once we had selected the most feasible attractant to the ‘transport’ for our experiments, we needed to know what could be the largest concentration of MgSO <sub>4</sub> in order to optimize the attraction of the nematode.
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- | <br/>
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- | To choose the concentration, were tested in a battery of increased concentrations regarding the initial medium (1 ml / L). Bearing in mind the results of
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- | the factor x2, we decided to test other factor concentrations: x3, x4, x5, x8 and x10; covering a range that does not exceed the concentration at which
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- | might affect the life of <i>C. elegans</i> or bacteria.
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- | <br/>
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- | Moreover, approaching experiments with <i>E. coli</i> and <i>Pseudomonas putida</i>, these trials were testing the end media: half plate with NGM
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- | non-altered and half as PHA production medium for <i>Pseudomonas</i> and interference of <i>E. coli</i>.
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- | <br/>
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- | Not knowing what fatty acid could activate transcription better, we tested two possibilities: oleic acid (named in the tables as PHAol) and octanoic acid
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- | (PHAoc). The concentration for each fatty acid was tested in <i>E. coli</i> choosing as better:
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- | <ul>
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- | <li>1.28 µl/ml of Octanoic acid.</li>
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- | <li>2.58 µl/ml of Oleic acid.</li>
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- | </ul>
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- | </p>
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- | <p>
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- | You can see our results in<b> figures 3, 4, 5 and 6</b>. The two first counts were made after 3 hours and the next ones after 6 hours. We suppose at that
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- | time the worms can select their “favorite” half part gone across and their movement would be always in the same area. We prepare as control plates with the
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- | same composition but without MgSO<sub>4</sub> in PHA media.
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- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/5/5b/Calling_table3.png" style="width:700px;" alt="Tables 3, 4, 5, 6" />
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- | </p>
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- | <p>
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- | <h3>Final concentration choice</h3>
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- | Once the test already performed and that the results seen by the factor of PHA and MgSO<sub>4</sub> selected fatty acid give very scattered (there is
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- | probably repellent effect at high concentrations in the medium with oleic but reversed in octanoic) we make a selection of MgSO<sub>4</sub> concentration
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- | for each medium.
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- | <ul>
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- | <li> <b>If it is used oleic acid </b><b>à</b><b> </b>Better results with 4ml/L MgSO<sub>4</sub>.
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- | </li>
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- | <li>
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- | <b>If octanoic acid used </b><b>à</b><b> </b>Best results to 10 ml/L of MgSO<sub>4</sub>.
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- | </li>
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- | </ul>
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- | Octanoic was discarded as a transcriptional activator of the iRNA of clumping after other result of <i>E. coli</i>, so finally we selected PHA medium with
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- | oleic acid.
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- | <br/>
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- | The biggest problem in trying to have an effective attractant was <b>how effective could be in presence of <i>E. coli</i></b>. It was therefore necessary
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- | to test the tradeoff between the value of 4ml / L MgSO<sub>4</sub> and pair it with different concentrations of bacteria, high enough to feed the nematode
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- | but low enough to permit the attractive effect of MgSO<sub>4</sub>.
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- | <br/>
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- | To find this point of commitment we prepare experiments in which the concentration of 4ml/L of MgSO<sub>4</sub> is faced against different ODs from serial
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- | dilutions of a preculture of E. coli DH5a.
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- | <br/>
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- | <b>Table 7 </b>
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- | shows the results. The count took place at 3h after the passing of fresh nematodes.
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- | </p>
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- | <p>
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- | <h3>Best E. coli OD choice</h3>
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- | An OD of 1 (minimum concentration of cells / volume) gives an attractive effect even better than expected (subsequent experiments try to see if there is
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- | synergy between <i>E. coli </i>and attractive factors MgSO4).
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- | <br/>
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- | To improve the approximation was decided to repeat the experiment with MgSO<sub>4</sub> concentration and the chosen bacteria OD showing that the system
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- | works well. The results can be seen in <b>figure 8.</b>
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- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/c/cc/Calling_table5.png" style="width:700px;" alt="Table 7" />
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- | </p>
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- |
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- |
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- | </div>
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- | <div>
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- | <h2 class="title"><img src="https://static.igem.org/mediawiki/2013/8/8e/White_clumping_icon.png" alt="" class="title-icon" />The Clumping</h2>
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- | <h1 class="title2 clumping-color"><img src="https://static.igem.org/mediawiki/2013/8/8e/White_clumping_icon.png" alt="" class="title2-icon clumping-bg" />The Clumping</h1>
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- |
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- | <p>
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- | The base of this part is our engineered <i>E.coli</i> with a biobrick that is capable of changing the worm’s behavior.
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- | </p>
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- | <p>
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- | Our<i> <b>C. elegans</b></i> strain <b>(N2 strain</b>) has two feeding behaviors: <b>Social and solitary feeding</b>. Social feeding is known as clumping
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- | as you can see in this video: <a href="http://www.youtube.com/watch?v=jCNsmcVVUVY">http://www.youtube.com/watch?v=jCNsmcVVUVY</a>
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- |
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- | </p>
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- | <p>
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- | Clumping is known to be induced under some conditions <b>like temperature or starving</b>, something that we had had considered during the entire project.
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- | However, it is also known how clumping is controlled from a genetic perspective and the main genes have already been described. We thought we could use
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- | this genetic approximation to control clumping under specific and controlled conditions. <i>(<a href="http://129.85.244.162/bard/pdf week_07_de_bono_bargmann_natural_variation_in_receptor_modifies_social_behavior_and_food_response_cell_1998.pdf
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- | ">view “</i>Natural variation in a neuropeptide Y receptor
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- | homolog modifies social behavior and food response in C. elegans.<b>”</b> Bono M, Bargmann CI</a>).
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- | </p>
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- | <p>
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- | <h3>How do we induce clumping?</h3>
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- | Our initial thought was to interfere the principal gene involved in this route: <b>NPR-1</b>. It is known that mutations in <b>NPR1</b> convert a solitary
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- | strain into a social strain. Another option we finally chose was <b>FLP-21</b>, a gene that positively regulates <b>NPR-1</b>. We selected this one instead
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- | because it is not involved in so many vital processes. <b>FLP-21</b> encodes a single FMRF amide-related neuropeptide that serves as a ligand for<b>Npr-1</b>, a G protein-coupled receptor that regulates social versus solitary feeding behavior in several <i>Caenorhabditis </i>species (see <b>fig. 1</b>).
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- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/0/04/Vb_clumping_1.png" alt="" style="width:700px" />
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- | </p>
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- | <p>
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- | So, this would be the final process: engineered<b> <i>E.coli</i> </b>that expresses <b>FLP-21</b>, whom <b>RNA will interfere </b>with the one codified by
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- | the worm because of the ingestion of bacteria. The formation of a complex of dsRNA will induce the elimination of <b>Flp-21 </b>transcripts by the RNA
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- | silencing pathway, which consequence is the induction of clumping (<b>see figure 2)</b>. We also had to take into account that the induction of clumping by
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- | this mechanism is almost immediate, in comparison with natural clumping which takes longer depending on the external factors.
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- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/f/f8/Vb_clumping_2.png" alt="" style="width:700px" />
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- | </p>
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- | <p>
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- | <h3>Why do we induce clumping for?</h3>
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- |
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- | The main objective of the synthetic symbiosis that we designed was to detect hotspots of interest in irregular substrates and the induction of clumping was a good tool for it. With this natural mechanism, which we were going to control, <b>worms kept in the desired places</b>, then <b>bacteria transported by <i>C. elegans </i></b>(<i>Pseudomonas putida)</i> <i> </i><b>were concentrated </b>where their action was needed, in order to generate value-added products such as bioplastic PHA and the increase of worms in a specific location <b>improved the image-based detection </b>mechanism (<a href="https://2013.igem.org/Team:Valencia_Biocampus/Devices">go to Devices section</a>)
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- | </p>
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- |
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- | <p>
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- | <h3>Controlling the mechanism</h3>
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- |
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- | The social feeding behavior needed to be controlled; it might be induced by conditions of interest as a tool for detecting hotspots in irregular substrates. We decided that <b>fatty acids </b>were going to be the inductors of the promoter (<b>fadBp</b>) that regulated the expression in the FLP-21
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- | antisense sequence, in order to induce RNA interference (<b>Fig.3)</b>. In the fatty acid rich media where this mechanism was activated, bacteria got off
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- | the worm, so this construction allows the induction of clumping in the spots where a biotechnological process will be carried out.
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- | </p>
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- |
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/9/93/Vb_clumping_3.png" alt="" style="width:700px" />
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- | </p>
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- |
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- | <p>
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- | <h3>Choosing the right fatty acid</h3>
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- | The <b><i>E. coli’s </i>biobrick<i> </i>promoter is induced by fatty acids</b>, but those also induce the promoter of <i>Pseudomonas putida </i>in order to
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- | produce PHA. Some investigations showed that the induction <b>of fadBp promoter </b>by <b>oleic acid </b>is the most efficient (<a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC216235/?page=1"><i>view “</i>Regulation of
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- | fatty acid degradation in Escherichia coli: analysis by operon fusion” Clark D. et al</a>) is
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- | higher when the fatty acid used is <b>oleic acid </b>but this is a long chained compound and that could affect the production of bioplastic (PHA). We
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- | decided to test the growth of <i>E. coli </i>and the production of bioplastic by <b><i>P. putida</i></b><i> </i>using PHA media with oleic acid and
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- | octanoic acid in order to get the best results in both activities.
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- | </p>
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- | <p>
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- | Both species of microorganisms where grown in PHA production media with different concentrations of oleic and octanoic acids, maintaining a global
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- | concentration of fatty acids of 8mM, because it was the one where we found the highest growth of both <i>E. coli </i>and <i>P. putida. </i> <b>Table 2 </b>
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- | (below) summarizes the assays done.
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- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/d/d1/Vb_clumping_4.png" alt="" style="width:700px" />
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- | </p>
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- | <p>
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- | The results of this assay showed that <i>E. coli </i>growth was better on PHA production media + 8mM oleic acid and the <i>P. putida </i>production of
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- | bioplastic was acceptable in this conditions. However, the highest production of bioplastic was on PHA production media+ 8mM octanoic acid one. So then, we
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- | made a <b>Colonization assay </b>and finally we determined that we were going to continue working using the PHA production media + 8mM oleic acid. Also,
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- | the ODs for plating both organisms were stablished as a consequence of this experiment. The “<b>Choosing the right fatty acid” </b>and the “<b>Colonization” </b>assays are explained at <a href="#4" data-liquidslider-ref="slider-id"><b>Building the bioplastic </b>section.</a> </b>
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- | </p>
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- |
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- | <p>
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- | <h3> Observing clumping induced by fatty acid rich media </h3>
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- |
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- | We could see how clumping was done induced by the presence of <b>8mM of oleic acid </b>(2.58 µl/ml ) which regulated the <b>FadBp promoter </b>of <b><i>E. coli</i></b><i> </i>and as a consequence the expression the complementary sequence to the worms’ <b>FLP-21 </b>transcript, producing interference
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- | by RNA. In the plates there was a high concentration of <i>E. coli </i>from the serial centrifugations preculture 4ml <b>XL1-Blue</b>, 2 min at maximum rpm
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- | (2 times). The following images show the change of behavior of <i>C. elegans, </i>which at first were eating alone and then they were doing it in groups as
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- | it was planned <b>(Fig. 4-6).</b>
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- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/4/4d/Vb_clumping_5.png" alt="" style="width:700px" />
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- | </p>
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- |
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- |
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- | </div>
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- | <div>
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- | <h2 class="title"><img src="https://static.igem.org/mediawiki/2013/9/97/White_building_icon.png" alt="" class="title-icon" />The Building</h2>
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- | <h1 class="title2 building-color"><img src="https://static.igem.org/mediawiki/2013/9/97/White_building_icon.png" alt="" class="title2-icon building-bg" />The Building</h1>
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- |
| |
- | <p>
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- | We decided to include in the mechanism design a regulated production of a value-added product in the hotspots of interest were C. elegans would be
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- | attracted to. We got proffit of the natural capacity of Pseudomonas putida of producing bioplastic PHA (polyhydroxyalkanoates) by bacterial fermentation of
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- | sugar or lipids (fatty acids in this case), because we found that the directed production of this versatile material was interesting due to its easy
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- | detection (complexes of PHA with red Nile are fluorescent), its bright future in the field of biomaterials and because we were using this natural ability
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- | of bacteria as a tool.
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- | </p>
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- |
| |
- | <p>
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- | <h3>The mechanism</h3>
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- |
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- | A cluster of genes is responsible of the production of PHA, the <b>phaC operon </b>of <b><i>P. putida </i></b>which consists of 4 ORFs that are transcribed
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- | in the same direction: phaC1 and phaC2 genes, codify for two PHA sintases; phaZ gene, codifies for a despolimerase; and phaD gene, that codifies for a
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- | protein of the TetR family. In the opposite direction two genes that codify for fasinas (phaF and phaI) and structural proteins are found <b>(Fig.1). </b>
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- | </p>
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- |
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/f/f8/Vb_building_1.png" alt="" style="width:700px" />
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- | </p>
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- |
| |
- | <p>
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- | <h3>The role of the operon in our project</h3>
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- |
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- | Our 2013 project is a proof of concept of the benefits that an artificial synthetic symbiosis between bacteria and nematodes can offer. The roles <i>Pseudomonas</i> play are two: firstly, they have been engineered to “ride” on <i>C. elegans</i> by the formation of a biofilm, but not stuffed with that, under the promoter glnA we stimulated the production of PHA, a value-added product that can be widely applied, for example:
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- | <ul>
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- | <li>
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- | For short disposable packaging items (personal hygiene products, surgical clothes).
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- | </li>
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- | <li>
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- | In upholstery.
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- | </li>
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- | <li>
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- | In the photographic and printing industry
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- | </li>
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- | <li>
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- | For textile industry since PHA can be processed into fibers.
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- | </li>
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- | <li>
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- | For biofuel production from PHA obtained from sewage sludge. This would combine two major advantages: the wastewater treatment and the generation of
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- | energy.
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- | </li>
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- | </ul>
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- | </p>
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- | <p>
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- | <h3>Choosing the right media</h3>
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- |
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- | On the plates we were going to have both <i>P. putida </i>and <i>E. coli </i>and their promoters’ response towards fatty acids was different, so we had to
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- | make a media where <b><i>E. coli </i></b>could induce<b> clumping </b>and <b><i>P. putida </i></b>the <b>production of bioplastic</b><i>. </i>In <b><i>E. coli, </i></b>the<b><i> </i></b>briobrick promoter for the expression of <b>FLP-21 iRNA </b>engineered in order to induce the social feeding
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- | behavior of<i> C. elegans </i><b>(Clumping) </b><b>is induced by fatty acids</b>, but not all the fatty acids are able to induce the same level expression
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- | (<b>Fig. 2</b>); <b>oleic acid </b>produces the highest activation of the promoter.
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- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/f/f1/Vb_building_2.png" alt="" style="width:700px" />
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- | </p>
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- |
| |
- | <p>
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- | Whereas the <b>standard medium </b>for the <b>production of PHA </b>by <b><i>Pseudomonas putida</i> </b>(composition reflected in <b>Fig.3</b>) employs <b>octanoic acid </b>(a short-chain fatty acid) as inducer. This fact made necessary to check if the modification of the fatty acid used, octanoic by
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- | oleic, would modify the bioplastic (PHA) production.
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- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/0/0d/Vb_building_3.png" alt="" style="width:700px" />
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- | </p>
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- | <p>
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- | Otherwise, the medium for PHA production is tuned for the development of <i>Pseudomonas putida</i>, but not for the <i>E.coli</i>, so we had to test if
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- | this bacteria was able to grow in it. When we did this checking, we realized that the development of these bacteria did not occur in the PHA production
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- | media. However, with the addition of acetate (carbon source), the removal of the iron salt (added to the media to avoid the synthesis of a <span class="siderophore-tooltip">siderophore</span> by <i>Pseudomonas sp.</i>) and, due to serendipity, with the double
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- | concentration of trace elementes, we got the media for the enteric bacteria growth.
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- | </p>
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- |
| |
- | <p>
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- | <h3>Choosing the right fatty acid</h3>
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- | Firstly, we tried to grow <i>E. coli </i>in the new PHA production media with different concentrations of octanoic acid. The same assays were done in
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- | parallel with <i>P. putida </i>in order to check which were the conditions where both microorganisms could grow and <i>P. putida </i>could produce enough
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- | quantity of bioplastic.
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- | </p>
| |
- | <p>
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- | After overnight incubation, the cultures were washed using sterile PBS and ODs<sub>600</sub> were measured (<b>Fig.4.</b>). We also <b>checked the PHA production </b>by centrifuging the precultures, resuspending the pellet on PBS and adding Red Nile dissolved in DMSO, we could see the
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- | emission of fluorescence due to the interaction between the Nile red and the PHA through an UV transilluminator <b>(Fig.5)</b>. By the results of the
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- | assays we chose to work with a <b>8mM</b> concentration of fatty acids (<b>Fig.4</b>).
| |
- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/d/dc/Vb_building_4.png" alt="" style="width:700px" />
| |
- | </p>
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- | <p>
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- | We also <b>checked the PHA production </b>by centrifuging the precultures, resuspending the pellet on PBS and adding Red Nile dissolved in DMSO, we could
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- | see the emission of fluorescence due to the interaction between the Nile red and the PHA through an UV transilluminator <b>(Fig.5)</b>. By the results of
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- | the assays we chose to work with a <b>8mM</b> concentration of fatty acids (<b>Fig.4</b>).
| |
- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/c/c9/Vb_building_5.png" alt="" style="width:700px" />
| |
- | </p>
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- | <p>
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- | At this point we found that <i>E. coli </i>and <i>P. putida </i>could grow at 8mM of octanoic acid and that bioplastic (PHA) is also produced. However,
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- | because <i>E. coli </i>grows better in <b>oleic acid </b>and we wanted to check if <b><i>P. putida </i></b>could produce PHA if its phaC operon was induced
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- | by it, we grew both bacteria in <b>different concentrations of octanoic and oleic acid but maintaining a constant concentration of fatty acids of 8mM</b>.
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- | The assays done are explained in the table below (<b>Fig.6</b>).
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- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/6/6a/Vb_building_6.png" alt="" style="width:700px" />
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- | </p>
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- | <p>
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- | We saw that <i>E. coli </i>had grown better with the increasing of oleic acid, getting at its maximum on experiment 9, even so, the quantity of pellet
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- | corresponding to 1 mL of preculture was scarce (and not visible at experiments 1 to 5). <i>E. coli </i>needed more time to grow in the PHA production
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- | media.
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- | </p>
| |
- | <p>
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- | <i>P. Putida</i>
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- | had grown too, however we wanted to measure the production of bioplastic, we measured the ODs<sub>600</sub> from the precultures of <i>P. putida </i>by
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- | centrifugating 2 mL of them and then washing them on 1 mL of PBS and resuspending the pellet in 500 μ<b>l </b>of PBS<b>. </b> Then, dilutions on PBS were
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- | adjusted to the lowest OD<sub>600</sub><sub>,</sub>corresponding to the experiment number 9 (8mM oleic acid). The growth of <i>P. putida </i>lower in PHA
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- | production media with a concentration of 8mM of oleic acid (<b>Fig. 7-8)</b>.
| |
- | </p>
| |
- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/9/9c/Vb_building_7.png" alt="" style="width:700px" />
| |
- | </p>
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- |
| |
- | <p>
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- | The results showed that the growth of <b><i>E. coli </i></b>on 8mM of oleic acid was better than in any other condition, then <b>experiment 9 </b>
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- | conditions would be the best ones for the induction of <b>clumping. </b>However, even <b><i>P. putida </i></b>had produced bioplastic at all oleic acid
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- | concentrations, the highest production levels corresponded to <b>experiment 1 </b>conditions, which means in absence of oleic acid, just on 8mM of octanoic
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- | acid. Because of that, next assays were done following the conditions of <b>experiment 1 and 9</b> (<b>Fig.9</b>).
| |
- | </p>
| |
- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/2/2f/Vb_building_8.png" alt="" style="width:700px" />
| |
- | </p>
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- | <p>
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- | After the optimization of the media, kinetics of the production of PHA and biomass generation were realized in relation to the time (<b>Fig.10</b>).
| |
- | </p>
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- | <p style="text-align:center">
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- | <img src="https://static.igem.org/mediawiki/2013/8/8c/Vb_building_9.png" alt="" style="width:700px" />
| |
- | </p>
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- |
| |
- | </div>
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- |
| |
- |
| |
- | <div>
| |
- | <h2 class="title"><img src="https://static.igem.org/mediawiki/2013/2/20/White_finalresult_icon.png" alt="" class="title-icon" />Pre-field test</h2>
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- | <h1 class="title2 finalresult-color"><img src="https://static.igem.org/mediawiki/2013/2/20/White_finalresult_icon.png" alt="" class="title2-icon finalresult-bg" />Pre-field test</h1>
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- |
| |
- | <p>
| |
- | <h3>Final Test: PHA Production in an Heterogeneous Substrate (soil)</h3>
| |
- | Finally, we designed an assay in which all the activities presented till this moment were carried out by the synthetic symbiosis that we have been working
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- | with. In this experiment we achieve the goal of our project<b>: the detection of hotspots of interest in irregular substrates and the production of bioplastic (PHA) in those places</b>. <u>Our project works in a pre-field test!</u>
| |
- | </p>
| |
- | <p>
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- | A soil ground was prepared with an artificially distributed substrate-rich medium in discrete points (<b>Fig.1</b>). Once the medium was ready, we
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- | inoculated the soil with a <b><i>C. elegans-Pseudomonas </i></b>suspension and with transformed <b><i>E. coli </i></b>expressing the iRNA of interest. As a
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- | control, we took a picture of the soil ground the first day the microorganisms were added by exposing it to UV light (<b>Fig.2</b>). After a day we added
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- | Nile red to reveal the PHA production and to see if the experiment had finally worked (<b>Fig. 3</b>). It is possible to see bioplastic nodules which are
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- | red when combined with Nile red.
| |
- | </p>
| |
- | <p style="text-align:center">
| |
- | <img src="https://static.igem.org/mediawiki/2013/d/d0/Vb_finalresult_1.png" alt="" style="width:700px" />
| |
- | </p>
| |
- | <p>
| |
- | In addition, we took an electron microscopy image of one of the discrete points of medium (<b>Fig. 4</b>) to study what organisms were actually near the
| |
- | hotspots. More electron microscopy images were taken to demonstrate if <i>C. elegans</i> dragged <i>Pseudomonas</i> to the points of interest ( <b>Fig. 4)</b>.
| |
- | </p>
| |
- |
| |
- | <p style="text-align:center">
| |
- | <img src="https://static.igem.org/mediawiki/2013/d/d6/Vb_finalresult_2.png" alt="" style="width:700px" />
| |
- | </p>
| |
- |
| |
- | <p>
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- | We finally were able to conclude that our synthetic consortium worked: Worms managed to reach the hotspots of interest, the worms dragged <i>Pseudomonas </i>with them and <i>Pseudomonas</i> finally made bioplastic detected be adding Nile red.
| |
- | </p>
| |
- | <p>
| |
- | In addition, we took an electron microscopy image of one of the discrete points of medium (<b>Fig. 4</b>) to study what organisms were actually near the
| |
- | hotspots. More electron microscopy images were taken to demonstrate if <i>C. elegans</i> dragged <i>Pseudomonas</i> to the points of interest ( <b>Fig. 4 [right] </b>).
| |
- | </p>
| |
- |
| |
- |
| |
- |
| |
- |
| |
- | </div>
| |
- | </div>
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