Team:Toulouse/Result/experimental-results

From 2014.igem.org

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<p class="title2">1. Petri dishes Test</p>
<p class="title2">1. Petri dishes Test</p>
<p class="texte">
<p class="texte">
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The first chemotaxis assay was done in Petri dishes filled with a growth medium containing 0,3% agar. This semi-solid medium was supposed to favor bacterial motility. A paper disk soaked with the attractive compound was placed in the middle of the dish, then cells were loaded into the medium (see Figure 1). This protocol was from the <a href="http://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>.
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The first chemotaxis assay was done in Petri dishes filled with a growth medium containing 0,3% agar. This semi-solid medium was supposed to favor bacterial motility. A paper disk soaked with the attractive compound was placed in the middle of the dish, then cells were loaded into the medium (see Figure 1). This protocol was adapted from <a href="http://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>.
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<center><img SRC="http://2014.igem.org/wiki/images/0/05/Schema_1.png" alt="schema Figure 1" style="width:500px"></center>
<center><img SRC="http://2014.igem.org/wiki/images/0/05/Schema_1.png" alt="schema Figure 1" style="width:500px"></center>
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<p class="legend">Figure 1: Petri dishes chemotaxis assay. (A) pipetman was used to inject cells into the semi-solid medium. (B) Bacteria would move toward the attractive compound diffusing from a paper disk.
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<p class="legend">Figure 1: Petri dishes chemotaxis assay. (A) A pipet was used to inject cells into the semi-solid medium. (B) Bacteria would move toward the attractive compound diffusing from a paper disk.
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<p class="texte">With this assay, we however failed to see any chemotaxis of the <i>wild-type</i> control toward glucose (Figure 2-A).As <i>B. subtilis</i> is attracted by many other glucides and amino-acids that can be found in rich medium such as in LB medium. Therefore, with the hope of improving the experimental conditions, we have challenged the cells with paper discs soaked in LB medium containing glucose (Figure 2-B).
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<p class="texte">With this assay, we however failed to see any chemotaxis of the <i>wild-type</i> control toward glucose (Figure 2-A). As <i>B. subtilis</i> is attracted by many other glucides and amino-acids that can be found in rich medium such as in LB medium. Therefore, with the hope of improving the experimental conditions, we have challenged the cells with paper discs soaked in LB medium containing glucose (Figure 2-B).
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<p class="texte">
<p class="texte">
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This protocol was from a PhD work (see references [1]). <i>B.subtilis</i> was grown overnight to a density of 8.10<sup>8</sup> cells/mL. 10mL of the culture was mixed with 15mL of LB medium containing 1.5% agar kept at 45°C. The final agar concentration was 0.9%. Tetracycline (25 µg/ml) was added to inhibit growth in order to only observe the chemotaxis phenomenon. Plates were cooled down and dried, before digging wells with either a punch or 1mL tips. The wells were then filled with the attractive compound (Figure 3). After one hour at room temperature, pictures of the plates were taken and the results analyzed.
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This protocol was adapted from a PhD work (see references [1]). <i>B.subtilis</i> was grown overnight to a density of 8.10<sup>8</sup> cells/mL. 10 mL of the culture was mixed with 15 mL of LB medium containing 1.5% agar kept at 45°C. The final agar concentration was 0.9%. Tetracycline (25 µg/ml) was added to inhibit growth in order to only observe the chemotaxis phenomenon. Plates were cooled down and dried, before digging wells with either a punch or 1mL tips. The wells were then filled with the attractive compound (Figure 3). After one hour at room temperature, pictures of the plates were taken and the results analyzed.
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<p class="texte">
<p class="texte">
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On our first try with <i>B. subtilis</i>, we made three wells per plate (Figure 4).The wells were filled with glucose at different concentrations and tetracycline was not added in one of the plates.
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On our first try with <i>B. subtilis</i>, we made three wells per plate (Figure 4). The wells were filled with glucose at different concentrations and tetracycline was not added in one of the plates.
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<p class="texte">
<p class="texte">
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After one hour, no tangible results were obtained. After 12h we observed halos around the 1M glucose containing wells in the plates without tetracycline but not in the plates with tetracycline. Again, because cells could use glucose for growth, we could not distinguish between growth and chemotaxis. Making the hypothesis that the concentration of tetracycline could have been too high and inhibited any bacterial activity, we thereafter lowered the tetracycline concentration to 15µg/mL. We repeated this protocol with this new experimental condition. We made two wells per plate (Figure 5), one with either Glucose or n-acetyl-glucosamine and one with LB medium. As previously, no results were achieved after 1h, but after 12h we could notice halos.
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After one hour, no tangible results were obtained. After 12h we observed halos around the 1 M glucose containing wells in the plates without tetracycline but not in the plates with tetracycline. Again, because cells could use glucose for growth, we could not distinguish between growth and chemotaxis. Making the hypothesis that the concentration of tetracycline could have been too high and inhibited any bacterial activity, we thereafter lowered the tetracycline concentration to 15 µg/mL. We repeated this protocol with this new experimental condition. We made two wells per plate (Figure 5), one with either Glucose or n-acetyl-glucosamine and one with LB medium. As previously, no results were achieved after 1h, but after 12h we could notice halos.
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<center><img SRC="http://2014.igem.org/wiki/images/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center>
<center><img SRC="http://2014.igem.org/wiki/images/c/c3/Bsubtilis_result.png" alt="Figure 5" style="width:750px"></center>
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<p class="legend">Figure 5: Chemotaxis test with <i>B. subtilis</i> WT. The upper well contains attractive compound and the lower well contains medium without any attractive compound.
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<p class="legend">Figure 5: Chemotaxis test with WT <i>B. subtilis</i>. The upper well contains attractive compound and the lower well contains medium without any attractive compound.
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<p class="texte">
<p class="texte">
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Results were not as clear as in the previous assay (Figure 4), but halos around the wells with glucose at 250mM with and without tetracycline were observed. With N-acetyl-glucosamine (NAG) as the attractive compound, halos were observed for a concentration of 25mM with tetracycline and for a concentration of 250mM without tetracycline, thus suggesting that our <i>B. subtilis</i> 168 strain is attracted toward NAG and uses it to grow.
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Results were not as clear as in the previous assay (Figure 4), but halos around the wells with glucose at 250 mM with and without tetracycline were observed. With N-acetyl-glucosamine (NAG) as the attractive compound, halos were observed for a concentration of 25 mM with tetracycline and for a concentration of 250 mM without tetracycline, thus suggesting that our <i>B. subtilis</i> 168 strain is attracted toward NAG and uses it to grow.
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<p class="title2">4. Capillary test between two tubes also called the tubes test</p>
<p class="title2">4. Capillary test between two tubes also called the tubes test</p>
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<p class="texte">After the experiment of the plug-in-pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p>
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<p class="texte">After the experiment of the plug-in-pond, we decided to construct a system by welding two Eppendorf tubes with a glass capillary and using an electric burner.</p>
<center><img src="http://2014.igem.org/wiki/images/f/fb/Chemotaxis_-_eppendorf.png"></center>
<center><img src="http://2014.igem.org/wiki/images/f/fb/Chemotaxis_-_eppendorf.png"></center>
<p class="legend">Figure 6: Photography of the first tubes system</p>
<p class="legend">Figure 6: Photography of the first tubes system</p>
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<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin dye towards water. However this construction had a leakage next to the weld seam that we could not stop.  
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<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin dye towards water. However this construction had a leakage next to the welding seam that we could not stop.  
Thus, the Toulouse 2014 iGEM team asked the help from the INSA glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p>
Thus, the Toulouse 2014 iGEM team asked the help from the INSA glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p>
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<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement. As we did not find any information in the litterature and did not have enough time to optimize this protocol, we dicided to test again the first protocol from the Imperial college 2011 iGEM team : the tips capillary test.
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<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement. As we did not find any information in the litterature and did not have enough time to optimize this protocol, we decided to test again and possibly ameliorate the first protocol from the Imperial college 2011 iGEM team : the tips capillary test.
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<br>
<br>
In the first tips capillary system, we used parafilm to avoid any kind of air disturbance in the tips. The different steps are described below:<br>
In the first tips capillary system, we used parafilm to avoid any kind of air disturbance in the tips. The different steps are described below:<br>
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- 15µL of each chemo-attractant was pipetted.<br>
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- 15 µL of each chemo-attractant was pipetted.<br>
- The bottom of tip with the pipette was then put on a piece of parafilm and the pipette was removed from the top of the tips.<br>
- The bottom of tip with the pipette was then put on a piece of parafilm and the pipette was removed from the top of the tips.<br>
- The tip was then sealed with a piece of parafilm in order to keep the liquid sterile and inside the tip.<br>
- The tip was then sealed with a piece of parafilm in order to keep the liquid sterile and inside the tip.<br>
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<p class="legend">Figure 9: Sealing of a tip with parafilm</p>
<p class="legend">Figure 9: Sealing of a tip with parafilm</p>
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<p class="texte">- When all the chemo-attractants were added, the were fixed on a green support. The whole process can be seen on Figure 10.<br>
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<p class="texte">- When all the chemo-attractants were added, the tips were fixed on a green support. The whole process can be seen on Figure 10.<br>
- Each tip was then immersed into 300 µL of a bacterial suspension in the wells of an Elisa plate.<br></p>
- Each tip was then immersed into 300 µL of a bacterial suspension in the wells of an Elisa plate.<br></p>
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<p class="texte"><i>NB: the yellow carton was used to stabilize the system and kept it straight.</i><br>
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and kept it straight.</i><br>
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<br>
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- After one hour, the tips were removed from the bacteria suspensions and the bacteria content of the tips was monitored with a Thoma cell under the microscope.<br>
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- After one hour, the tips were removed from the bacteria suspensions and the bacteria content within the tips was monitored with a Thoma cell under the microscope.<br>
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<br>
We experienced several problems with this system:<br>
We experienced several problems with this system:<br>
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- The liquid level decreasing so much during the course of the experiment that we did not have enough liquid to fill the Thoma cell for counting.<br>
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- The liquid level decreasing so badly during the course of the experiment that we did not have enough liquid to fill the Thoma cell for counting.<br>
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- The bacteria were moving, therefore preventing us from accurately counting them. Taking into account this probelem, we decided to estimate the bacterial concentration by streaking the tips content on agar plate instead of using Thoma cell and microscopy.
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- The bacteria were moving, therefore preventing us from accurately counting them (but that's the point of swimming, no??? ;-)). Taking into account this problem, we decided to estimate the bacterial concentration by streaking the tips content on agar plate instead of using Thoma cell and microscopy.
<p class="title3">Second tips capillary system
<p class="title3">Second tips capillary system
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The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than that of the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br>
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolality bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than that of the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br>
<br>
<br>
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At the beginning, the experiment was conducted with only one negative control, the fuchsin and different NAG concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis</i> 168:<br>
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At the beginning, the experiment was conducted with only one negative control, the fuchsin and different NAG concentrations: 25 mM, 250 mM and 500 mM. The tested strain was <i>Bacillus subtilis</i> 168:<br>
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<center>
<center>
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However, our team did not give up on synthetic biology! :-) Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br>
However, our team did not give up on synthetic biology! :-) Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br>
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<br>
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Hopefully, we managed to find a negative control: galactose (25mM). The article "Chemotaxis towards sugars by <i>B. subtilis</i>" (<i>Ordal et al., 1979</i>) proved that it was a poor attractant.<br>
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Hopefully, we managed to find a negative control: galactose (25 mM). The article "Chemotaxis towards sugars by <i>B. subtilis</i>" (<i>Ordal et al., 1979</i>) proved that it was a poor attractant.<br>
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<br>
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We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p>
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We made our tests again with this new molecule and glucose (25 mM) as positive control.<br></p>
<center><img src="http://2014.igem.org/wiki/images/8/86/Chemotaxis_-_final_results.png"></center>
<center><img src="http://2014.igem.org/wiki/images/8/86/Chemotaxis_-_final_results.png"></center>
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<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br>
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br>
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<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p>
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<b><p class="texte">Our results are not statistically significant however this result has been described in the litterature.</p></b><br></p>
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<p class="title2">1. Preliminary experiments</p>
<p class="title2">1. Preliminary experiments</p>
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<p class="texte">For the first experiment we wanted to check if the buffer of the binding assay was compatible with <i>B. subtilis</i> survival. To do that, we tested four bacterial concentrations (from OD 0.1 to OD 0.01). These <i>B. subtilis</i> suspensions were incubated 1 hour at 4°C with 500µL of either Chitin Binding Buffer (CBB) or water. 100 µL cell suspension were plated on LB medium in order to count surviving cells.</br>
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<p class="texte">For the first experiment we wanted to check if the buffer of the binding assay was compatible with <i>B. subtilis</i> survival. To do that, we tested four bacterial concentrations (from OD 0.1 to OD 0.01). These <i>B. subtilis</i> suspensions were incubated 1 hour at 4°C with 500 µL of either Chitin Binding Buffer (CBB) or water. 100 µL of cell suspension were plated on LB medium in order to count surviving cells.</br>
Cells do not seem affected by the presence of CBB or water with estimated ODs of 0.05 or 0.025.
Cells do not seem affected by the presence of CBB or water with estimated ODs of 0.05 or 0.025.
</br>We observed similar survival rates between cells treated with CBB or water (these data are not shown on figure 16).
</br>We observed similar survival rates between cells treated with CBB or water (these data are not shown on figure 16).
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Both bacterial solutions of WT and engineered bacterium used for the binding assay had the initial same concentration before the assay (Direct on Figure 17).
Both bacterial solutions of WT and engineered bacterium used for the binding assay had the initial same concentration before the assay (Direct on Figure 17).
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There is also no significant difference between both strains after the first wash (Wash A on Figure 17). However, the concentration of cells quantified in the eluted fraction after the second wash was significantly higher for the wild-type strain. This suggests that the engineered strain is more retained on chitin beads. This was confirmed by a 40 times higher number of cells associated with the chitin beads for the engineered over the wild-type strain(Beads on Figure 17).  
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There is also no significant difference between both strains after the first wash (Wash A on Figure 17). However, the concentration of cells quantified in the eluted fraction after the second wash was significantly higher for the wild-type strain. This suggests that the engineered strain is more retained on chitin beads. This was confirmed by a 40 times higher number of cells associated with the chitin beads for the engineered over the wild-type strain (Beads on Figure 17).  
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<br/>Thus, we successfully engineered <i>B. subtilis</i> to promote its fixation on chitin.</p>
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<br/>Thus, we successfully engineered <i>B. subtilis</i> to promote its binding onto chitin.</p>
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<p class="legend">Figure 18: Microscopic view of engineered strain associated with beads surfaces coated with chitin</p>
<p class="legend">Figure 18: Microscopic view of engineered strain associated with beads surfaces coated with chitin</p>
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<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments.</br></p>
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<p class="texte">Using ImageJ software, we created 3D pictures and movies of those images.</br></p>
<center><img src="http://2014.igem.org/wiki/images/5/53/Photo_billes_microscopie.png" width="45%" style="float:left;"><video width="45%" poster="http://2014.igem.org/wiki/images/4/4c/Video_poster.png" controls>
<center><img src="http://2014.igem.org/wiki/images/5/53/Photo_billes_microscopie.png" width="45%" style="float:left;"><video width="45%" poster="http://2014.igem.org/wiki/images/4/4c/Video_poster.png" controls>
<source src="http://2014.igem.org/wiki/images/6/61/Beads_3D_movie.ogg" type='video/ogg; codecs="theora, vorbis"'/>
<source src="http://2014.igem.org/wiki/images/6/61/Beads_3D_movie.ogg" type='video/ogg; codecs="theora, vorbis"'/>
</video></center>
</video></center>
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<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin and the engineered strain (emotional sequence for Subtitree: first movie apparition, before Cannes…)</p>
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<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin and the engineered strain (emotional sequence for Subtitree: first movie apparition, before Cannes festival…)</p>
<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 (Right on Figure 20), there is less release than without the module (Right on Figure 20). Therefore, the engineered bacterium is retained by the beads.</p>
<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 (Right on Figure 20), there is less release than without the module (Right on Figure 20). Therefore, the engineered bacterium is retained by the beads.</p>
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<p class="title2"> 1. Preliminary experiments</p>
<p class="title2"> 1. Preliminary experiments</p>
<p class="title3">Tests with commercial peptides and controls</p>
<p class="title3">Tests with commercial peptides and controls</p>
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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). As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus. These tests were therefore performed with different non-pathogenic fungal strains from the same phylum as <i>Ceratocystis Platani</i>.
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<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5 µM, 25 µM, 100 µM). As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus. These tests were therefore performed with different non-pathogenic fungal strains from the same phylum as <i>Ceratocystis Platani</i>.
</br>
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After 1 to 6 days at 30°C depending on the fungal strain, the PDA (Potato Dextrose Agar) plates covered with fungus and containing a paper disk soaked with a commercial peptides solution were analyzed.</p>
After 1 to 6 days at 30°C depending on the fungal strain, the PDA (Potato Dextrose Agar) plates covered with fungus and containing a paper disk soaked with a commercial peptides solution were analyzed.</p>
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<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i> (Figure 21). Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions. Copper Sulfate, a well-known chemical fungicide was used as a positive control.Inhibition of the fungal growth was complete with 20 mg/ml copper Sulfate, and at 10 mg/ml a darker halo appeared around the pad as can be seen on figure 21. This corresponds to a sporulating halo in response to the stress generated by the fungicide.
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<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100 µM on <i>Aspergillus brasiliensis</i> (Figure 21). Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions. Copper sulfate, a well-known chemical fungicide was used as a positive control. Inhibition of the fungal growth was complete with 20 mg/ml copper sulfate, and at 10 mg/ml a darker halo appeared around the pad as can be seen on Figure 21. This corresponds to a sporulating halo in response to the stress generated by the fungicide.
</p>
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<p class="texte">Regarding these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth in the tested conditions. Following these tests, new conditions were adopted in order to avoid too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees, and a 'sap-like' medium was elaborated (See <a href="http://2014.igem.org/Team:Toulouse/Notebook/Protocols">protocols</a> for more informations). Incubations were performed at room temperature. These new conditions were used as standard for the the next experiment.<br>
<p class="texte">Regarding these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth in the tested conditions. Following these tests, new conditions were adopted in order to avoid too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees, and a 'sap-like' medium was elaborated (See <a href="http://2014.igem.org/Team:Toulouse/Notebook/Protocols">protocols</a> for more informations). Incubations were performed at room temperature. These new conditions were used as standard for the the next experiment.<br>
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Camille also concluded that turning blue the Canal du Midi using high copper sulfate concentrations is not such a good idea... Thereby strengthening our faith in SubtiTree :-) !  
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Camille also concluded that turning the water of the Canal du Midi to deep blue using high copper sulfate concentrations is not such a good idea... Thereby strengthening our faith in SubtiTree :-) !  
</p>
</p>
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<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves could be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants do not have deleterious effects.</br>
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves could be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants do not have deleterious effects.</br>
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Without proper treatment, the drop of the phytopathogenic fungus on <i>Nicotiana benthamiana</i> leaves caused a necrosis halo which could be measured after 40h. The  number of necrotic sites and the lesion size appeared as reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike the WT bacterium. Two independant replicate of this experiments were performed successfully</br><br></br>
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Without proper treatment, the drop of the phytopathogenic fungus on <i>Nicotiana benthamiana</i> leaves caused a necrosis halo which could be measured after 40 h. The  number of necrotic sites and the lesion sizes appeared reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike the WT bacterium. Two independant replicate of this experiments were performed successfully</br><br></br>
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br>
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br>
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<p class="texte">We could expect that bringing altogether the three modules (chemotaxis, binding and antifungal) should even improved the performance of SubtiTree. Thus, these results open the way towards the use of SubtiTree in plane tree</br>
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<p class="texte">We now expect that bringing altogether the three modules (chemotaxis, binding and antifungal) should even improve the performance of SubtiTree. Thus, these results pave the way towards the testing of SubtiTree in infected plane trees</br>
More than ever, let's save our trees with SubtiTree!
More than ever, let's save our trees with SubtiTree!
</p>
</p>

Revision as of 01:11, 18 October 2014