Team:Toulouse/Result/experimental-results

From 2014.igem.org

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<p class="texte"><We could not notice any difference between the petri dish with or without glucose on paper. With an addition of LB medium to sugar, a large halo around paper disk is observed. This halo may corresponds to cells attracted by solution, or it may be diffusion of the mix.</br>
<p class="texte"><We could not notice any difference between the petri dish with or without glucose on paper. With an addition of LB medium to sugar, a large halo around paper disk is observed. This halo may corresponds to cells attracted by solution, or it may be diffusion of the mix.</br>
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Anyway we did not have enough reproduccible and reliable results to be satisfied with this test. Furthermore if we are forced to add LB to sugar to observe something, it is hard to distinguish between attracting and chemotaxis effects.</br>
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Anyway we did not have enough reproducible and reliable results to be satisfied with this test. Furthermore if we are forced to add LB to sugar to observe something, it is hard to distinguish between attracting and chemotaxis effects.</br>
We have started new tries using different protocols</p>
We have started new tries using different protocols</p>
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This protocol on which we worked is taken from a thesis (ref thèse). B.subtilis are grown overnight and if necessary bacteria cells are concentrated by centrifgation. Goal is to obtain a cells density to 8x10⁸ cells/mL. 10mL of bacteria cells are mixed with 15mL of LB medium with 1.5 % agar maintained at 50°C. We obtain a medium with 0.9 % agar at final concentration. We add tetracyclin at 25µg/mL thus growth are stopped. Plate are cooled and dryed, then well are made with punch or 1mL tips. In well attractive compound are put (Figure 3). After one hour at room temperature, we take a picture of plates and analysed results.
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This protocol on which we worked is taken from a thesis (ref thèse). <i>B.subtilis</i> are grown overnight and if necessary bacteria cells are concentrated by centrifugation. Goal is to obtain a cells density to 8x10⁸ cells/mL. 10mL of bacteria cells are mixed with 15mL of LB medium with 1.5 % agar maintained at 50°C. We obtain a medium with 0.9 % agar at final concentration. We add tetracycline at 25µg/mL thus growth are stopped. Plate are cooled and dried, then well are made with punch or 1mL tips. In well attractive compound are put (Figure 3). After one hour at room temperature, we take a picture of plates and analyzed results.
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On our first try with bacillus subtilis, we made three wells by plate (Figure 4).In wells we put glucose at different concentration and in one of the plate we do not put tetracyclin.
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On our first try with <i>B. subtilis</i>, we made three wells by plate (Figure 4). In wells we put glucose at different concentration and in one of the plate we do not put tetracycline.
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We respect the protocol and after one hour we observe nothing, it's only after 12h than we can observe an halo around well with glucose at 1M in the plate where there are no tetracyclin. Tetracyclin concentration seems to be too large and inhibit our bacteria. Thereafter we have work with tetracyclin at 15µg/mL.
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We respect the protocol and after one hour we observe nothing, it's only after 12h than we can observe an halo around well with glucose at 1M in the plate where there are no tetracycline. Tetracycline concentration seems to be too large and inhibit our bacteria. Thereafter we have work with tetracycline at 15µg/mL.
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We retry this protocol with less tetracyclin. We made two wells by plate (Figure 5) one with attractive compound, Glucose or n-acetyl-glucosamide and one with LB medium. After 1h there are no halos, 12h after we observe something.
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We retry this protocol with less tetracycline. We made two wells by plate (Figure 5) one with attractive compound, Glucose or n-acetyl-glucosamide and one with LB medium. After 1h there are no halos, 12h after we observe something.
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<center><img SRC="https://static.igem.org/mediawiki/2014/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center>
<center><img SRC="https://static.igem.org/mediawiki/2014/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center>
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<p class="legend">Figure 5: Chemotaxis test with Bacillus subtilis WT. The upper well contain attractive compound and the lower contain medium without attractive compound. </p>
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<p class="legend">Figure 5: Chemotaxis test with <i>Bacillus subtilis</i> WT. The upper well contain attractive compound and the lower contain medium without attractive compound. </p>
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Results are not as clear as the first time, but we observe halo around well with glucose at 250mM with and without tetracyclin. We have made tries with N-acetyl-glucosamide and we see no halo, this show that our strain Bacillus subtilis 168 is not attracted by N-acetyl-glucosamide.</br>
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Results are not as clear as the first time, but we observe halo around well with glucose at 250mM with and without tetracycline. We have made tries with N-acetyl-glucosamide and we see no halo, this show that our strain <i>B. subtilis</i> 168 is not attracted by N-acetyl-glucosamide.</br>
Results are not enough clear and reliable with plug-in-pond. We do not understand why we have to wait 12 hours to see halos. So we tried other protocols.
Results are not enough clear and reliable with plug-in-pond. We do not understand why we have to wait 12 hours to see halos. So we tried other protocols.
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<b>References:</b></br>
<b>References:</b></br>
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thesis : Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez Escherichia coli et Shewanella oneidensis, 2008, Claudine Baraquet, université de la méditerranée Aix-Marseille II
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thesis : Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez Escherichia coli et Shewanella oneidensis, 2008, Claudine Baraquet, université de la méditerranée Aix-Marseille II
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Revision as of 21:01, 15 October 2014