Team:Toulouse/Result/experimental-results

From 2014.igem.org

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<p class="texte">
<p class="texte">
<b>References:</b></br>
<b>References:</b></br>
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[1] : 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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[1]: Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez <i>Escherichia coli</i> et <i>Shewanella oneidensis</i>, 2008, Claudine Baraquet, Université de la Méditerranée Aix-Marseille II
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<p class="title2">1. Preliminary experiments</p>
<p class="title2">1. Preliminary experiments</p>
<p class="title3">Purpose</p>
<p class="title3">Purpose</p>
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<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the CBB buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p>
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<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the Chitin Beads Buffer (CBB) buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p>
<p class="title3">Results</p>
<p class="title3">Results</p>
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<p class="texte">The bacterial solutions could not be counted because of two major problems: the too high number of bacteria with the 0.1 OD or the too low number of bacteria with the 0.01 OD. Thus, the study is mostly focused on the intermediate values (Figure 16).
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<p class="texte">We could not count bacteria either because of the high number of bacteria with the 0.1 OD solution or the low number of bacteria with the 0.01 OD solution. Thus, the study is mostly focused on the intermediate values (Figure 16).
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<br/>First of all, a same cell concentration can be noticed with the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which agrees with the dilution ratio.  
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<br/>First of all, cells do not seem affected by the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio.
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</br>Thus the the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C are compatible with the bacterial life.  
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.
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<p class="legend">Figure 16: CBB presence has no effect on bacteria. The bacterial concentration was measured regarding  <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span>of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).
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<p class="legend">Figure 16: CBB presence has no effect on bacterial survival. The bacterial concentration was measured in <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span> of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).</p>
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<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p>
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p>
<p class="title3">Purpose</p>
<p class="title3">Purpose</p>
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<p class="texte">Transformed <i>Bacillus subtilis</i> with the binding module is able to produce a protein composed of the bacterial peptidoglycan bonding of LycT and the GbpA 4th domain of <i>Vibrio cholerae</i> allowing the chitin bonding. The synthetic bacterium is put with special beads composed of the polymer miming the fungal pathogen wall. After several washes, bacteria specifically attached to the chitin are put on plates and counted.</p>
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<p class="texte"><i>B. subtilis</i> transformed with the binding module should produce a chimeric protein composed of the Cell Wall Binding Domain of LycT to attach the chimeric protein to the cell wall, and the GbpA domain 4 of <i>Vibrio cholerae</i> to bind chitin. The synthetic bacterium is put in contact with chitin beads (chitin: polymer present on the fungal pathogen wall). After several washes, bacteria remaining on the beads are counted.</p>
<p class="title3">Results</p>
<p class="title3">Results</p>
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<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration : 105 bacteria/mL (Figure 17). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more Wild Type bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module.   
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<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration: 10^5 bacteria/mL (Figure 17). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it removes 40 times more wild-type bacteria than SubtiTree. This result correlates to the number of Subtitree bound to the beads.   
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<br/>Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.</p>
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<br/>Thus, the binding system is validated: SubtiTree binds efficiently to chitin.</p>
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<p class="legend">Figure 17:   Attachment of <i>Bacillus subtilis</i> with binding module to chitin. <span style="color:#0000FF">The WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentration has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.</p>
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<p class="legend">Figure 17: Attachment of WT <i>B. subtilis</i> and Subtitree the to chitin. The <span style="color:#0000FF">WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentrations have been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.
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<p class="title2">3. Microscopic observations</p>
<p class="title2">3. Microscopic observations</p>
<p class="title3">Purpose</p>
<p class="title3">Purpose</p>
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<p class="texte">We want to observe the SubtiTree's binding on beads coated with chitin. In order to perform a 3D reconstruction showing this interaction, we use confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we can highlight the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determined the minimum threshold to remove the background noise and the natural fluorescence.</p>
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<p class="texte">We wanted to observe SubtiTree bound on the chitin coated beads. In order to perform a 3D reconstruction showing this interaction, we used confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we highlighted the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determined the minimum threshold to remove the background noise and the natural fluorescence.</p>
<p class="title3">Results</p>
<p class="title3">Results</p>
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<p class="texte">First, we note the great bacterial presence on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p>
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<p class="texte">First, we can notice that SubtiTree is sitting (well, sort off!!) on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p>
<center><img src="http://2014.igem.org/wiki/images/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center>
<center><img src="http://2014.igem.org/wiki/images/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center>
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<center><img src="http://2014.igem.org/wiki/images/5/53/Photo_billes_microscopie.png" width="45%" style="float:left;"><iframe width="380" height="315" src="//www.youtube.com/embed/ztIHIKQr3g0" frameborder="0" allowfullscreen></iframe></center>
<center><img src="http://2014.igem.org/wiki/images/5/53/Photo_billes_microscopie.png" width="45%" style="float:left;"><iframe width="380" height="315" src="//www.youtube.com/embed/ztIHIKQr3g0" frameborder="0" allowfullscreen></iframe></center>
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<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin</p>
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<p class="legend">A short movie of 3D bead surfaces coated with chitin and Subtitree (emotional sequence for Subtitree: first movie apparition, before Cannes…)</p>
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<p class="texte">Finally we want to observe the bacteria after the second wash. When our bacterium has the binding module, results suggest a lower number of bacteria in the washing solution. SubtiTree is retained by the beads.</p>
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<p class="texte">We then performed a wash step on the chitin beads. We measured the release of bacteria on the washing solution. When our bacterium has the binding module, there is less release and therefore, SubtiTree is retained by the beads.</p>
<center><img src="http://2014.igem.org/wiki/images/9/97/Photo_lavage_microscopie.png" width="45%"></center>
<center><img src="http://2014.igem.org/wiki/images/9/97/Photo_lavage_microscopie.png" width="45%"></center>
<p class="legend">Figure 20: Microscopic view of bacteria after washing  
<p class="legend">Figure 20: Microscopic view of bacteria after washing  
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<p class="texte">Finally, overall results are consistent with the presence of functional binding system.</p>
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<p class="texte">Finally, all results are consistent with the presence of functional binding system. We thus validate the second module.</p>
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Revision as of 21:01, 16 October 2014