Team:Toulouse/Result/experimental-results

From 2014.igem.org

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<p class="title2">3. Tips capillary system
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<p class="title2">3. Tips capillary system</p>
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<p class="title3">First tips capillary system</p>
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<p class="title3">First tips capillary system
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<p class="texte">This protocol comes from Imperial College iGEM team 2011 and was adapted by our team in several steps (See <a href="http://2014.igem.org/Team:Toulouse/Notebook/Protocols">chemotaxis protocol</a>).<br>
<p class="texte">This protocol comes from Imperial College iGEM team 2011 and was adapted by our team in several steps (See <a href="http://2014.igem.org/Team:Toulouse/Notebook/Protocols">chemotaxis protocol</a>).<br>
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<p class="legend">Figure 6 : Second tips capillary system</p>
<p class="legend">Figure 6 : Second tips capillary system</p>
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<p class="title3">Improvement of the second tips capillary system
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<p class="title3">Improvement of the second tips capillary system</p>
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<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p>
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p>
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<p class="title1">Binding module
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<p class="title1">Binding module</p>  
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<p class="title2"> 1. Preliminary experiments</p>
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<p class="title2"> 1. Preliminary experiments
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<p class="title3">Purpose</p>
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<p class="title3">Purpose
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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>
<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="title3">Results
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<p class="title3">Results</p>
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<p class="texte">The bacterial solutions could not be counted because of two main 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 1).
<p class="texte">The bacterial solutions could not be counted because of two main 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 1).
<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 is in agreement with the dilution ratio.  
<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 is in agreement with the dilution ratio.  
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<p class="title2">2. Binding test using engineered <i>B. subtilis</i>
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<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p>
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<p class="title3">Purpose
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<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">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.
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<p class="title3">Results
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<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 2). 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.   
<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 2). 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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<br/>Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.
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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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<p class="texte">Figure 2:  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.
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<p class="texte">Figure 2:  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="title2">3. Microscopic observations
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<p class="title2">3. Microscopic observations</p>
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<p class="title3">Purpose
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<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 determine the minimum threshold to remove the background noise and the natural fluorescence.</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 determine the minimum threshold to remove the background noise and the natural fluorescence.
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<p class="title3">Results
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<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>
<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>
<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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<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.</br>
<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.</br>
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Finally, overall results are consistent with the presence of functional binding system.
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Finally, overall results are consistent with the presence of functional binding system.</p>
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<p class="title1">Fungicides module
<p class="title1">Fungicides module
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<p class="title2"> 1. Preliminary experiments
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<p class="title2"> 1. Preliminary experiments</p>
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<p class="title3">Tests with commercial peptides and controls</p>
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<p class="title3">Tests with commercial peptides and controls
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<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12,5µM/25µM/100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12,5µM/25µM/100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br>
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br>
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After several days at 30°C, the PDA (Potato Dextrose Agar) plates couvered with fungus and commercial peptides were analyzed.
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After several days at 30°C, the PDA (Potato Dextrose Agar) plates couvered with fungus and commercial peptides were analyzed.</p></p>
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<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions.As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below.  This corresponds to a sporing halo in response to the stress generated by the fungicide.
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions.As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below.  This corresponds to a sporing halo in response to the stress generated by the fungicide.
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<p class="title2">2. Test with SubtiTree
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<p class="title2">2. Test with SubtiTree<p/>
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<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br>
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br>
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<p class="title2">3. <i> In planta </i> tests with SubtiTree
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<p class="title2">3. <i> In planta </i> tests with SubtiTree<p/>
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Revision as of 11:29, 13 October 2014