Team:Toulouse/Result/experimental-results

From 2014.igem.org

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<p class="texte"> We used and developed several protocols to demonstrate the existence of chemotaxis in <i>B. subtilis</i> wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine.  
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<p class="texte">We performed several assays to demonstrate the chemotaxis ability of our engineered <i>Bacillus subtilis</i> strain to move towards N-acetylglucosamine (NAG), the base unit of chitin. The ability of the <i>wild-type</i> strain to move towards glucose was the positive control.  
</p>
</p>
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<p class="title2">1. Petri Dishes Test </p>
 
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<p class="texte">We first wanted to visualize chemotaxis on Petri dishes. We hoped to obtain pictures with bacteria halos directed or around attractive components and thus tried different protocols. The first protocol was adapted from the one published by <a href="http://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a>. They put the attractive compound on a paper disk in the middle of a Petri dish containing a medium with 0.3% agar. Cells are loaded in this medium (Figure 1).
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<p class="title2">1. Petri dishes Test</p>
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<p class="texte">
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</br>
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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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<b>We first tried to test chemotaxis onto Petri Dishes filled with a 0.3% agar medium. This semi-solid medium allows the bacterial motility. A paper disk containing an attractive compound is placed in the middle of the dish and cells are then loaded in the medium (see Figure 1). This protocol was taken from the <a href="http://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 2011 iGEM team</a></b>.</p>
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</p>
<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: Scheme showing how cells are filled into the medium. (A) A pipet tip is used to deposit cells in the gelose. (B) Bacteria should move toward the attractive compound which diffuses.</p>
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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>
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<p class="texte">We did not have any result with WT <i>Bacillus subtilis</i> and glucose as attractive compound (Figure 2-A). <i>B. subtilis</i> is attracted by many other glucides and amino-acids, so we also tried to test diluted glucose in LB medium attractant (Figure 2-B).</p>
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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>
<center><img SRC="http://2014.igem.org/wiki/images/f/ff/Fig2_AetB.png" alt="Figure 2" style="width:750px"></center>
<center><img SRC="http://2014.igem.org/wiki/images/f/ff/Fig2_AetB.png" alt="Figure 2" style="width:750px"></center>
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<p class="legend">Figure 2: Chemotaxis test with Glucose as attractive compound (A) and Glucose added to LB medium as attractant (B).</p>
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<p class="legend">Figure 2: Chemotaxis test with paper discs soaked in either a Glucose solution (A) or in Glucose containing LB medium (B) as attractive compound. Paper discs soaked with water were used as negative controls.
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</p>
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<p class="texte"> We could not notice any difference between the petri dish with or without glucose. With an addition of LB medium to sugar, a large halo around the paper disk was noticeable. This halo may correspond to cells attracted by the solution, as it is not noticeable when cells are not added (data not shown). Anyway we did not have enough reproducible and reliable results to be satisfied with this test.<br>
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<p class="texte">No difference was observed between the Petri dishes with or without glucose. With glucose containing LB medium, a large halo around the paper disk was observed. This halo might be due to cells attracted by the solution, as it was not observed when cells were not inoculated in the Petri dish (data not shown). However this result was barely  reproducible. Moreover with the addition of LB medium, it was hard to make the distinction between attractive effects and cell growth resulting from random diffusion. We have therefore given up this protocol and tested alternative protocols.
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Furthermore, with the addition of LB medium, it is hard to make the distinction between the attractive effects and the simple growth resulting from random diffusion.</br>
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</p>
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We have started new tries using different protocols.</p>
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<p class="title2">2. Plug in Pond system
<p class="title2">2. Plug in Pond system
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<p class="texte">
<p class="texte">
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This protocol we worked on is taken from a thesis (see references [1]). . A solution of <i>B.subtilis</i> is grown overnight so as to obtain a cell density of 8x10⁸ cells/mL. 10mL of the solution is mixed with 15mL of LB medium with 1.5 % agar kept at 45°.The final concentration of the obtained medium is 0.9% agar. Tetracycline is aded at 25µg/mL, in order to inhibit growth and to only observe the chemotaxis phenomenon. Plates are cooled and dried, before digging wells with a punch or 1mL tips. The wells are filled with attractive compounds (Figure 3). After one hour at room temperature, photos of the plates are taken and the results are analyzed.
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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.
</p>
</p>
<center><img SRC="http://2014.igem.org/wiki/images/c/cd/Fig3.png" alt="Figure 3" style="width:400px"></center>
<center><img SRC="http://2014.igem.org/wiki/images/c/cd/Fig3.png" alt="Figure 3" style="width:400px"></center>
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<p class="legend">Figure 3: Schema showing how are made plug-in-pond tests.</p>
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<p class="legend">Figure 3: Plug-in-pond test design.</p>
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<p class="texte">
<p class="texte">
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After an hour, no tangible results were obtained. It is only after 12hours that we were able to observe halos around the wells with glucose at 1M in the plates without tetracycline. Tetracycline concentration seems to be too high and inhibits any bacterial activity. Therfore, we have worked with tetracycline at 15µg/mL.
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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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We tried this protocol again with this new condition. We made two wells per plate (Figure 5), one with either Glucose or N-acetyl-glucosamine and one with LB medium. As previsously, no results were achieved after 1h, but after 12hours we could notice halos.
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</p>
</p>
<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>Bacillus subtilis</i> WT. The upper wells 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>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>
<p class="texte">
<p class="texte">
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Results are not as clear as the first time, but we observed halos around the well with glucose at 250mM with and without tetracycline. We have then tried the same experiment with N-acetyl-glucosamine and we did not see any halo in the tested conditions. Thus we assumed that our <i>B. subtilis</i> 168 strain was not attracted to N-acetyl-glucosamine.
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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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However, the results are not clear, reliable and reproducible enough with the plug-in-pond protocol. Another testing protocol was then adopted.  
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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 <i>Escherichia coli</i> et <i>Shewanella oneidensis</i>, 2008, Claudine Baraquet, Université de la Méditerranée Aix-Marseille II
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[1]: Claudine Baraquet. <b>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></b>. 2008, Université de la Méditerranée Aix-Marseille II.
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<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 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 weld seam that we could not stop.  
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Thus, we 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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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="legend">Figure 7: Scheme of the tubes system</p>
<p class="legend">Figure 7: Scheme of the tubes system</p>
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<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT <i>Bacillus subtilis</i> and N-Acetylglucosamine.
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<p class="texte">
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This new system was tested with the fuchsin dye and the assay was made with WT <i>B. subtilis</i> and N-Acetylglucosamine as the attractive compound.
<br><br>
<br><br>
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<i>NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of the small diameter of the capillary.  
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<i>NB: We could not see any diffusion of the fushin dye from one tube to the other. We made the hypothesis that it was not visible by sight because of the small diameter of the capillary.  
</i><br>
</i><br>
<br>
<br>
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br>
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br>
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- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.<br>
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- The first step was the addition of Wash Buffer until the capillary was full to prevent the presence of any air bubbles which could have impeded diffusion.<br>
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- Then, the tube 2 was plugged with the thumb while another person was adding the bacterial solution of WT <i>Bacillus subtilis</i> in the tube 1. <br>
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- Then, the tube 2 was plugged and maintained with the thumb while another iGEM mate was adding the bacterial suspention of WT <i>B. subtilis</i> into the tube 1.<br>
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- The tube 1 was also plugged and only after the thumb could be removed from the tube 2. <br>
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- The tube 1 was also plugged and only then the thumb could be removed from the tube 2. <br>
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- In the same way, the N-Acetylglucosamine was added in the tube 2. <br>
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- In the same way, the N-Acetylglucosamine was added in the tube 2.<br>
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- The same process was made with a xylose positive control.<br>
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- The same process was made with xylose as a positive control.<br>
<br>
<br>
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<i>NB: According to the article Chemotaxis towards sugars by </i>Bacillus subtilis, (George W. Ordal et al., 1979), <i>glucose and xylose have the same attractant power. We prefer a positive control instead of a negative one because we were not sure that this system was efficient.</i><br>
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<i>NB: According to the paper "Chemotaxis towards sugars by </i>Bacillus subtilis", (Ordal et al., 1979),<i> glucose and xylose have the same attractant power. We have privileged a positive control instead of a negative one as we were not sure that this system was efficient.</i><br>
<br>
<br>
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- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).</p>
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- The system was kept straight for 2 hours. Every 40 minutes, samples from each were removed and streaked on solid medium (dilution 1/1,000) in order to estimate the bacterial concentration.</p>
<center><img src="http://2014.igem.org/wiki/images/1/1b/Chemotaxis_-_tubes_photo.png"></center>
<center><img src="http://2014.igem.org/wiki/images/1/1b/Chemotaxis_-_tubes_photo.png"></center>
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<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.<br>
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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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As we did not have the time to optimize this protocol we preferred using the protocol of the 2011 Imperial college iGEM team : the tips capillary test.</br>
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</p>
</p>
<p class="title2"> 5. Tips capillary system</p>
<p class="title2"> 5. Tips capillary system</p>
<p class="title3">First tips capillary system</p>
<p class="title3">First tips capillary system</p>
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<p class="texte">This protocol comes from 2011 Imperial College iGEM team and was adapted by our team in several steps (See <a href="http://2014.igem.org/Team:Toulouse/Notebook/Protocols#select8">chemotaxis protocol</a>).
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<p class="texte">This protocol from Imperial College 2011 iGEM team was adapted by our team in several steps (See <a href="http://2014.igem.org/Team:Toulouse/Notebook/Protocols#select8">chemotaxis protocol</a>).
<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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- 15µL of each chemo-attractant was pipetted.<br>
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- 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 tip.<br>
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- 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>
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- The top of the tip was then sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip. <br>
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- The tip was then sealed with a piece of parafilm in order to keep the liquid sterile and inside the tip.<br>
- To finish, the level of the solution in the tip was marked.<br></p>
- To finish, the level of the solution in the tip was marked.<br></p>
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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">- After all the attractants were added in the tips, we put them on a green base to carry them. 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 were fixed on a green support. The whole process can be seen on Figure 10.<br>
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- Each tip was immersed in 300 µL of a bacterial solution in the wells of an Elisa plate.<br></p>
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- Each tip was then immersed into 300 µL of a bacterial suspension in the wells of an Elisa plate.<br></p>
<center><img src="http://2014.igem.org/wiki/images/0/05/Chemotaxis_-_tip_and_support.png"></center>
<center><img src="http://2014.igem.org/wiki/images/0/05/Chemotaxis_-_tip_and_support.png"></center>
<p class="legend">Figure 10: First tips capillary system</p>
<p class="legend">Figure 10: First tips capillary system</p>
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<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep it straight.</i><br>
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<p class="texte"><i>NB: the yellow carton was used to stabilize the system and kept it straight.</i><br>
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- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed 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 of the tips was monitored with a Thoma cell under the microscope.<br>
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<br>
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We had several problems with this system:<br>
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We experienced several problems with this system:<br>
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- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.<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 bacteria were moving and therefore, we could not proceed to a bacteria count.<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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<br>
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Regarding these observations we decided to spread the tips content on agar plates instead of using Thoma cell and microscopy.<br>
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<p class="title3">Second tips capillary system
<p class="title3">Second tips capillary system
</p>
</p>
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<p class="texte"And then the revolution came! We found a multichannel pipette :D The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p>
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<p class="texte">And then the revolution came! We found a multichannel pipette :D The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p>
<center><img src="http://2014.igem.org/wiki/images/e/e4/Chemotaxis_-_pipette.png"></center>
<center><img src="http://2014.igem.org/wiki/images/e/e4/Chemotaxis_-_pipette.png"></center>
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<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br>
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br>
<br>
<br>
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There was just one tiny problem… we did not have our optimized bacterium transformed with the chemotaxis module!!! That is why we concentrated our efforts on WT <i>Bacillus subtilis</i> strain.<br>
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There was just one tiny problem… we did not have our optimized bacterium transformed with the chemotaxis module!!! That is why we concentrated our efforts on WT <i>B. subtilis</i> strain.<br>
<br>
<br>
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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 the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br>
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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>
<br>
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At the beginning, the experiment was conducted with only one negative contraol, 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: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis</i> 168:<br>
<br></p>
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<center>
<center>
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<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr>
<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr>
</table></center><br>
</table></center><br>
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<p class="texte">The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.<br>
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<p class="texte">The average number of colonies with the negative control is 121. In contrast, a cell layer was observed for the NAG plates with every concentration, indicating that a large number of cells have been inoculated in the plates.<br>
<br>
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Thus, we assumed that WT <i>Bacillus subtilis</i> was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons. <br>
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Thus, we assumed that WT <i>B. subtilis</i> was more attracted by NAG than by fuchsin. In addition we could neglect the bacterial growth because the test only lasted one hour. We also neglected diffusion and osmolality phenomena for the reasons explained above. <br>
<br>
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Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.<br>
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Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells and thus make the fuschin dye an appropriate negative control.<br>
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<b><p class="texte">This incredible and dramatic discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br>
<b><p class="texte">This incredible and dramatic discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br>
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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>
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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>
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Hopefully, we managed to find a negative control: galactose (25mM). The article Chemotaxis towards sugars by <i>Bacillus subtilis</i> (<i>George W. 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 (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>
<br>
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We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p>
We made our tests again with this new molecule and glucose (25mM) as positive control.<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="title3">Purpose</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">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>
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Cells do not seem affected by the presence of CBB or water with estimated ODs of 0.05 or 0.025.
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</br>We observed similar survival rates between cells treated with CBB or water (data not shown).
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<p class="title3">Results</p>
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Thus, the experimental conditions of the chitin binding assay are compatible with the bacterial life.</p>
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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, 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.  
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<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>
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</br>
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<center><img src="http://2014.igem.org/wiki/images/c/ce/Graphe_binding_1.png" width="45%"></center>
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<center><img src="http://2014.igem.org/wiki/images/e/ea/Graphe_binding_2.png" width="60%"></center>
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+
</br>
</br>
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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>
+
 
 +
<p class="legend">Figure 16</p>
<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>
-
<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="texte"><i>B. subtilis</i> transformed with the binding module should produce a chimeric protein allowing the bacterium to bind chitin. It is composed of the Cell Wall Binding Domain of a <i>B. subtilis</i> protein LycT, and of the GbpA domain 4 of <i>Vibrio cholerae</i> able to bind chitin. The capacity to bind chitin was assessed by bringing together either the WT strain or the engineered bacterium with chitin beads. After several washes, bacteria still bound to the beads were counted.</p>
<p class="title3">Results</p>
<p class="title3">Results</p>
-
<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<sup>5</sup> 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.
+
<p class="texte">We measured the quantity of bacteria before the binding assay (Direct), in the eluted fraction of the first (Wash A) and the second (Wash B) washing steps, and associated with the beads.</br>  
-
<br/>Thus, the binding system is validated: SubtiTree binds efficiently to chitin.</p>
+
 
 +
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).
 +
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).  
 +
<br/>Thus, we successfully engineered <i>B. subtilis</i> to promote its fixation on chitin.</p>
</br>
</br>
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</br>
</br>
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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.
+
<p class="legend">Figure 17: Attachment of WT <i>B. subtilis</i> and engineered bacterium 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.
</p>
</p>
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<p class="title3">Purpose</p>
<p class="title3">Purpose</p>
-
<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="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 green 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>
-
<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>
+
<p class="texte">We can notice the engineered bacterium is well attached to the surface of beads coated with chitin.</p>
 +
</br>
<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>
</br>
</br>
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<p class="legend">Figure 18: Microscopic view of beads surfaces coated with chitin</p>
+
<p class="legend">Figure 18: Microscopic view of engineered strain associated with beads surfaces coated with chitin</p>
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments.</br></p>
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments.</br></p>
<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>
</br>
</br>
-
<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin and Subtitree (emotional sequence for Subtitree: first movie apparition, before Cannes…)</p>
+
<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>
-
<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>
+
<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>
<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 elution fraction of WT <i>B. subtilis</i> (Left) and engineered bacterium (Right).
</p>
</p>
-
<p class="texte">Finally, all results are consistent with the presence of functional binding system. We thus validate the second module.</p>
+
<p class="texte">To conclude, all the results are consistent with the successfull integration of a functional chitin binding system in <i>B. subtilis</i>. We thus validated the second module.</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>
-
<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). 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>.
-
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br>
+
</br>
-
After several days at 30°C, the PDA (Potato Dextrose Agar) plates covered with fungus and commercial peptides were analyzed.</p></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>
-
<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 sporulating 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> (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>
</p>
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<center><img style="width:450px; " src="http://parts.igem.org/wiki/images/a/a8/Prelim_tests_fung.jpg">
<center><img style="width:450px; " src="http://parts.igem.org/wiki/images/a/a8/Prelim_tests_fung.jpg">
<img style="width:450px; " src="http://parts.igem.org/wiki/images/f/f8/Controls_fung.jpg"></center>
<img style="width:450px; " src="http://parts.igem.org/wiki/images/f/f8/Controls_fung.jpg"></center>
-
<p class="legend"> Figure 21: Results of the preliminary tests</p>
+
<p class="legend"> Figure 21: Results of the preliminary tests of antifungal compounds</p>
</br>
</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 not to encourage 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: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.<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>
-
We 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 :-) !  
+
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 :-) !  
</p>
</p>
-
<p class="title2">2. Test with SubtiTree</p>
+
<p class="title2">2. Test with antifungal bacteria</p>
-
<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 our <i>Bacillus subtilis</i> engineered strains, it was essential to find the right balance between the fungal growth and the bacterial one which was achieved with the previous modifications. Furthermore, in our genetic constructions, the antifungal peptides were designed to be exported in the extracellular medium.</br>
</br>
</br>
-
The transformed <i>Bacillus subtilis</i> strains grew at 37°C during 72h and were tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the GAFP-1 + D4E1 operon (see the photos below).</br>
+
The transformed <i>Bacillus subtilis</i> strains were grown at 37°C during 72h before testings. Inhibitory effect of supernatant and cell pellets were tested separately. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i> growth was clearly observable for the strain expressing the D4E1 peptide. The inhibition was even more noticeable with the strain carrying the GAFP-1 + D4E1 operon (Figure 22).</br>
-
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br>
+
However, no effect was detected for the strain expressing the GAFP-1 gene, we thus suppose a putative synergistic effect between these two peptides.</br>
-
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.
+
Regarding EcAMP-1, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP-1 and in addition, the sequencing results for these constructs showed several discrepancies with the original designed sequence.
-
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium.  
+
<p class="texte">Inhibition halos are not visible with supernatants, probably because of either low concentrations or of instability in the extracellular medium.  
-
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliensis</i>. This effect is comparable to the one previously noted with a low concentration of sulfate copper. </br>
+
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus, <i>Aspergillus brasiliensis</i> (figure 22). This effect is comparable to the one previously noted with a low concentration of copper sulfate  (figure 21).</br>
</p>
</p>
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<p class="texte">
<p class="texte">
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The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br>
+
After this set of experiments, the strains expressing D4E1 or GAFP-1 + D4E1 have been shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b>we decided to tests these strains in model plants</b>: <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br>
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After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants</b>: <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br>
+
These tests were performed in association with Sylvain Raffaële and Marielle Barascud in the National Institute for the Agronomic Research.</p>
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These tests were performed in the National Institute for the Agronomic Research by experts in this domain.  
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<center><img style="width:215px;" img src="http://parts.igem.org/wiki/images/a/af/In_planta.jpg" style="margin-top:5px"/></center>
<center><img style="width:215px;" img src="http://parts.igem.org/wiki/images/a/af/In_planta.jpg" style="margin-top:5px"/></center>
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<p class="legend"> Figure 23: Injection of SubtiTree in a model plant</p>
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<p class="legend"> Figure 23: Injection of antifungal <i>B. subtilis</i> in a model plant</p>
<p class="texte">
<p class="texte">
-
The goal of the project is to introduce the transformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br>
+
The goal of the project is to introduce the transformed bacteria in a diseased tree. So it is necessary to perform preliminary<i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous sets of experiments. </br>
-
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves.  </br>
+
The strain expressing fungicides was first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) was placed on the leaves. </br>
-
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br>
+
</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>
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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>
-
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can 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 pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem to be reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</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">Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.</p>
+
<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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More than ever, let's save our trees with SubtiTree!
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Revision as of 00:09, 18 October 2014