Team:Toulouse/Result/experimental-results

From 2014.igem.org

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<p class="texte"> We performed several tests to demonstrate the chemotaxis ability of transformed <i>Bacillus subtilis</i> towards NAG and we used WT bacteria and glucose as a positive glucose.  
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<p class="texte"> We performed several tests to demonstrate the chemotaxis ability of the transformed <i>Bacillus subtilis</i> strain towards NAG and we used the wild type bacteria and glucose as a positive glucose.  
</p>
</p>
<p class="title2">1. Petri Dishes Test </p>
<p class="title2">1. Petri Dishes Test </p>
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<p class="texte"> We first tried to test chemotaxis onto Petri Dishes filled with a medium containing 0,3% agar. This semi-solid medium is supposed to allow bacterial motility. A paper disk with 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>.</p>
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<p class="texte"> 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>.</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: Schema showing how cells are filed in the medium. (A) pipetman are used to put cells in the medium. (B) Bacteria should move to the attractive compound which diffuses.</p>
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<p class="legend">Figure 1: Schema showing how cells are filled in the medium. (A) Pipettes are used to put cells in the medium. (B) Bacteria should move to the attractive compound which diffuses.</p>
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<p class="texte">We did not have any result with <i>Bacillus subtilis</i> WT 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 have diluted glucose in LB mediums attractant (Figure 2-B).</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>
<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 in add to LB medium as attractant (B).</p>
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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>
<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>  
<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>  
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>
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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We have started new tries using different protocols</p>
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We have started new tries using different protocols.</p>
<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 on which we worked is taken from a thesis (ref thèse). 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 compound (Figure 3). After one hour at room temperature, the plates are caught in photos and the results are analyzed.
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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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<p class="texte">
<p class="texte">
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After an hour, no tangible results were achieved. It's only after 12h than 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. Thereafter we have worked with tetracycline at 15µg/mL.
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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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We retry this protocol 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 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.
</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 well contain attractive compound and the lower contain medium without attractive compound. </p>
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<p class="legend">Figure 5: Chemotaxis test with <i>Bacillus subtilis</i> WT. The upper wells contain attractive compound and the lower contain medium without attractive compound. </p>
<p class="texte">
<p class="texte">
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Results are not as clear as the first time, but we observe halo around the well with glucose at 250mM with and without tetracycline. We have made tries with N-acetyl-glucosamine and we did not see halo in the tested conditions, thus supposing that our strain <i>B. subtilis</i> 168 is not attracted by N-acetyl-glucosamine. MOI JE VOIS DES HALOS ??? </br>
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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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However, the results are not enough clear, reliable and reproducible with the plug-in-pond protocol. Another testing protocol was then adopted.  
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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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thesis : Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez Escherichia coli et Shewanella oneidensis, 2008, Claudine Baraquet, université de la méditerranée Aix-Marseille II
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[1] : Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez Escherichia coli et Shewanella oneidensis, 2008, Claudine Baraquet, université de la méditerranée Aix-Marseille II
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<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 capillary thanks to 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>
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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.  
<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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Thus, the Toulouse iGEM Team asked the help from the glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p>
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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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- The same process was made with a xylose positive control.<br>
- The same process was made with a xylose positive control.<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 because we were not sure that this system was efficient.</i><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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- 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>
- 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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<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>
<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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As we did not have the time to optimize this protocol we preferred using the protocol of the Imperial college iGEM team 2011: the tips capillary test.</br>
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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>
</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 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#select8">chemotaxis protocol</a>).<br> PRINCIPE DU TEST
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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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First of all, parafilm was used to close the tips:<br>
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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 then pipetted. <br>
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- 15µL of each chemo-attractant was pipetted. <br>
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- The tips with the pipette were then put on a piece of parafilm and the pipette was removed from 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 tip.<br>
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- The tip was 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 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>
- 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 chemo-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">- 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>
- Each tip was immersed in 300 µL of a bacterial solution in the wells of an Elisa plate.<br></p>
- Each tip was immersed in 300 µL of a bacterial solution 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 keep it straight.</i><br>
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep 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 Thoma cell under the microscope.<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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We had several problems with this system:<br>
We had several problems with this system:<br>
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- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br>
- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br>
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Regarding these observations we decided to spread the tips content on agar plate instead of using Thoma cell and microscopy.<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>
<p class="title3">Second tips capillary system
<p class="title3">Second tips capillary system
</p>
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<p class="texte"And then the revolution came! We found a multichannel pipette. 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>
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There was just one tiny problem… we did not have our optimized bacterium wtransformed 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>Bacillus subtilis</i> strain.<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>
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 experiment was conducted with fuchsin as a negative control and was tested with different positive controls: glucose (25mM) and xylose (25mM).<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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We obtained the following result with NAG at different concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis </i>168:<br>
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<br></p>
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<center>
<center>
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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>
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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<b><p class="texte">This incredible discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><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>
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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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We finally used galactose (25mM) as a negative control. 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>Bacillus subtilis</i> (<i>George W. Ordal et al., 1979</i>) proved that it was a poor attractant.<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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<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>
<p class="legend">Figure 15: Final results (dilution : 1/10,000)</p>
<p class="legend">Figure 15: Final results (dilution : 1/10,000)</p>
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<p class="texte"> The miracle arrived! We managed to prove that our WT Bacillus subtilis was indeed naturally attracted to NAG</p>
<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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Revision as of 19:19, 16 October 2014