Team:Toulouse/Result/experimental-results

From 2014.igem.org

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<p class="title2">2. Capillary test between two tubes also called the tubes test
<p class="title2">2. Capillary test between two tubes also called the tubes test
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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.  
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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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<center>
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<center><table align="center">
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<table align="center">
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<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></tr></td>
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></tr></td>
<tr><td align=center>Figure 1 : Photography of the first tubes system</tr></td>
<tr><td align=center>Figure 1 : Photography of the first tubes system</tr></td>
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</table><br>
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</table></center><br>
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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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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, 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.
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<center><table align="center">
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<center>
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<table align="center">
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<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"></tr></td>
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"></tr></td>
<tr><td align=center>Figure 2 : Scheme of the tubes system</tr></td>
<tr><td align=center>Figure 2 : Scheme of the tubes system</tr></td>
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</table></center><br>
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As we did previously, we tested this new system with fuchsin. This experiment was made with WT Bacillus subtilis  and N-Acetylglucosamine.
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<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT Bacillus subtilis  and N-Acetylglucosamine.
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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 by the small diameter of the capillary.  
<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 by the small diameter of the capillary.  
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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 Bacillus subtilis, (George W. Ordal et al., 1979), 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 Bacillus subtilis, (<i>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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- 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).
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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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<center><table align="center">
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<center>
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<table align="center">
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></tr></td>
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></tr></td>
<tr><td align=center>Figure 3 : Photography of the tubes system</tr></td>
<tr><td align=center>Figure 3 : Photography of the tubes system</tr></td>
</table></center><br>
</table></center><br>
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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 and the time was too short. We did not find any information in the literature.<br>
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>
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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<p class="title2">3. Tips capillary test
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<p class="title2">3. Tips capillary system
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<p class="title3">First tips capillary test
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<p class="title3">First tips capillary system
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<p class="texte">
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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="https://2014.igem.org/Team:Toulouse/Notebook/Protocols">chemotaxis protocol</a>).<br>
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First of all, parafilm was used to close the tips:<br>
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- 15µL of each chemo-attractant was then 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 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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- To finish, the level of the solution in the tip was marked.<br></p>
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<center>
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<table align="center">
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<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></tr></td>
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<tr><td align=center>Figure 4 : Sealing of a tip with parafilm</tr></td>
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</table></center><br>
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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 5.<br>
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- Each tip was put in 300 µL of a bacteria solution in the wells of an Elisa plate.<br></p>
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<center>
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<table align="center">
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<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></tr></td>
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<tr><td align=center>Figure 5 : First tips capillary system</tr></td>
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</table></center><br>
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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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- 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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We had 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 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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<p class="title3">Second tips capillary system
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</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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<center>
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<table align="center">
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<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></tr></td>
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<tr><td align=center>Figure 6 : Second tips capillary system</tr></td>
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</table></center><br>
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<p class="title3">Improvement of the second tips capillary system
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<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p>
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<center>
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<table align="center">
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<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></tr></td>
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<tr><td align=center>Figure 7 : Improvement of the second tips capillary system</tr></td>
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</table></center><br>
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<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 with the chemotaxis gene… That is why we concentrated our efforts on WT Bacillus subtilis  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 osmolarity 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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We obtained the following result with NAG at different concentrations: 25mM, 250mM and 500mM. The tested strain was Bacillus subtilis 168:<br>
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<center>
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<table align="center">
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<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td>
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<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr>
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<tr><td align=center>Figure 8 : Fuchsin - negative control (dilution 1/50)</td>
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<td align=center>Figure 9 : NAG (25mM) (dilution 1/50)</td></tr>
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</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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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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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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However, our team did not give up on synthetic biology and on our strength! 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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We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p>
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<center>
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<table align="center">
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<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></tr></td>
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<tr><td align=center>Figure 10 : Final results (dilution : 1/10,000)</tr></td>
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</table></center><br>
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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>
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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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Revision as of 21:39, 9 October 2014