Team:Toulouse/Result/experimental-results

From 2014.igem.org

(Difference between revisions)
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<p class="texte">We wanted to see chemotaxis on petri dish. We hoped to obtain pictures with bacteria halos directed or around attractive components. Thus we tried different protocols on <i>Bacillus subtilis</i>.</br>
<p class="texte">We wanted to see chemotaxis on petri dish. We hoped to obtain pictures with bacteria halos directed or around attractive components. Thus we tried different protocols on <i>Bacillus subtilis</i>.</br>
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The first one was a protocol from the Imperial College <a href="http://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">2011 iGEM team</a>. They put attractive compound on paper disk in the middle of a petri dish containing a medium with 0.3% agar. Cells are loaded in this medium (Fig 1).</p>
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The first one was a protocol from the Imperial College <a href="http://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">2011 iGEM team</a>. They put attractive compound on paper disk in the middle of a petri dish containing a medium with 0.3% agar. Cells are loaded in this medium (Figure 1).</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">Fig1 : schema showing how cells are filed in the medium. (A) pipetman are used to put cells in the gelose. (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 filed in the medium. (A) pipetman are used to put cells in the gelose. (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 positive test on <i>Bacillus subtilis</i> and with glucose as attractive compound (Fig 2-A). <i>B. sub</i> is attracted by many other glucides and amino-acids so we have diluted glucose in LB medium and used this solution as a target (Fig 2-B).</p>
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<p class="texte">We did not have any result with positive test on <i>Bacillus subtilis</i> and with glucose as attractive compound (Figure 2-A). <i>B. sub</i> is attracted by many other glucides and amino-acids so we have diluted glucose in LB medium and used this solution as a target (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">Fig 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 in add to LB medium as attractant (B).</p>
<p class="texte"><We could not notice any difference between the petri dish with or without glucose on paper. With an addition of LB medium to sugar, a large halo around paper disk is observed. This halo may corresponds to cells attracted by solution, or it may be diffusion of the mix.</br>
<p class="texte"><We could not notice any difference between the petri dish with or without glucose on paper. With an addition of LB medium to sugar, a large halo around paper disk is observed. This halo may corresponds to cells attracted by solution, or it may be diffusion of the mix.</br>
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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). B.subtilis are grown overnight and if necessary bacteria cells are concentrated by centrifgation. Goal is to obtain a cells density to 8x10⁸ cells/mL. 10mL of bacteria cells are mixed with 15mL of LB medium with 1.5 % agar maintained at 50°C. We obtain a medium with 0.9 % agar at final concentration. We add tetracyclin at 25µg/mL thus growth are stopped. Plate are cooled and dryed, then well are made with punch or 1mL tips. In well attractive compound are put (Fig3). After one hour at room temperature, we take a picture of plates and analysed results.
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This protocol on which we worked is taken from a thesis (ref thèse). B.subtilis are grown overnight and if necessary bacteria cells are concentrated by centrifgation. Goal is to obtain a cells density to 8x10⁸ cells/mL. 10mL of bacteria cells are mixed with 15mL of LB medium with 1.5 % agar maintained at 50°C. We obtain a medium with 0.9 % agar at final concentration. We add tetracyclin at 25µg/mL thus growth are stopped. Plate are cooled and dryed, then well are made with punch or 1mL tips. In well attractive compound are put (Figure 3). After one hour at room temperature, we take a picture of plates and analysed results.
</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">Fig 3 : schema showing how are made plug-in-pond tests.</p>
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<p class="legend">Figure 3: Schema showing how are made plug-in-pond tests.</p>
<p class="texte">
<p class="texte">
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On our first try with bacillus subtilis, we made three wells by plate (Fig4).In wells we put glucose at different concentration and in one of the plate we do not put tetracyclin.
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On our first try with bacillus subtilis, we made three wells by plate (Figure 4).In wells we put glucose at different concentration and in one of the plate we do not put tetracyclin.
</p>
</p>
<center><img SRC="http://2014.igem.org/wiki/images/c/ce/Fig4.png" alt="Figure 4" style="width:400px"></center>
<center><img SRC="http://2014.igem.org/wiki/images/c/ce/Fig4.png" alt="Figure 4" style="width:400px"></center>
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<p class="legend">Fig 4 : Plates after 12h at room temperature.</p>
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<p class="legend">Figure 4: Plates after 12h at room temperature.</p>
<p class="texte">
<p class="texte">
We respect the protocol and after one hour we observe nothing, it's only after 12h than we can observe an halo around well with glucose at 1M in the plate where there are no tetracyclin. Tetracyclin concentration seems to be too large and inhibit our bacteria. Thereafter we have work with tetracyclin at 15µg/mL.
We respect the protocol and after one hour we observe nothing, it's only after 12h than we can observe an halo around well with glucose at 1M in the plate where there are no tetracyclin. Tetracyclin concentration seems to be too large and inhibit our bacteria. Thereafter we have work with tetracyclin at 15µg/mL.
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We retry this protocol with less tetracyclin. We made two wells by plate (Fig5) one with attractive compound, Glucose or n-acetyl-glucosamide and one with LB medium. After 1h there are no halos, 12h after we observe something.
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We retry this protocol with less tetracyclin. We made two wells by plate (Figure 5) one with attractive compound, Glucose or n-acetyl-glucosamide and one with LB medium. After 1h there are no halos, 12h after we observe something.
</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 Bacillus subtilis 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 Bacillus subtilis WT. The upper well contain attractive compound and the lower contain medium without attractive compound. </p>
<p class="texte">
<p class="texte">
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<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="legend">Figure 1: Photography of the first tubes system</p>
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<p class="legend">Figure 6: Photography of the first tubes system</p>
<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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<center><img src="http://2014.igem.org/wiki/images/2/2b/Chemotaxis_-_tubes.png"><center>
<center><img src="http://2014.igem.org/wiki/images/2/2b/Chemotaxis_-_tubes.png"><center>
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<p class="legend">Figure 2: Scheme of the tubes system</p>
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<p class="legend">Figure 7: Scheme of the tubes system</p>
<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.
<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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<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="legend">Figure 3: Photography of the tubes system</p>
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<p class="legend">Figure 8: Photography of the tubes system</p>
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<center><img src="http://2014.igem.org/wiki/images/9/94/Chemotaxis_-_tip.png"></center>
<center><img src="http://2014.igem.org/wiki/images/9/94/Chemotaxis_-_tip.png"></center>
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<p class="legend">Figure 4: Sealing of a tip with parafilm</p>
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<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 5.<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 10.<br>
- Each tip was put in 300 µL of a bacteria solution in the wells of an Elisa plate.<br></p>
- Each tip was put in 300 µL of a bacteria solution 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>
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<p class="legend">Figure 5: First tips capillary system</p>
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<p class="legend">Figure 10: First tips capillary system</p>
<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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<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="legend">Figure 6: Second tips capillary system</p>
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<p class="legend">Figure 11: Second tips capillary system</p>
<p class="title3">Improvement of the second tips capillary system</p>
<p class="title3">Improvement of the second tips capillary system</p>
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<center><img src="http://2014.igem.org/wiki/images/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center>
<center><img src="http://2014.igem.org/wiki/images/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center>
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<p class="legend">Figure 7: Improvement of the second tips capillary system</p>
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<p class="legend">Figure 12: Improvement of the second tips capillary system</p>
<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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<tr><td align=center><img src="http://2014.igem.org/wiki/images/8/8c/Chemotaxis_-_results_fuch.png"></td>
<tr><td align=center><img src="http://2014.igem.org/wiki/images/8/8c/Chemotaxis_-_results_fuch.png"></td>
<td align=center><img src="http://2014.igem.org/wiki/images/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr>
<td align=center><img src="http://2014.igem.org/wiki/images/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr>
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<tr><td align=center><p class="legend">Figure 8: Fuchsin - negative control (dilution 1/50)</p></td>
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<tr><td align=center><p class="legend">Figure 13: Fuchsin - negative control (dilution 1/50)</p></td>
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<td align=center><p class="legend">Figure 9: NAG (25mM) (dilution 1/50)</p></td></tr>
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<td align=center><p class="legend">Figure 14: NAG (25mM) (dilution 1/50)</p></td></tr>
</table></center><br>
</table></center><br>
<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>
<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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<center><img src="http://2014.igem.org/wiki/images/8/86/Chemotaxis_-_final_results.png"></center>
<center><img src="http://2014.igem.org/wiki/images/8/86/Chemotaxis_-_final_results.png"></center>
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<p class="legend">Figure 10 : Final results (dilution : 1/10,000)</p>
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<p class="legend">Figure 15: Final results (dilution : 1/10,000)</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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<p class="title3">Results</p>
<p class="title3">Results</p>
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<p class="texte">The bacterial solutions could not be counted because of two main problems: the too high number of bacteria with the 0.1 OD or the too low number of bacteria with the 0.01 OD. Thus, the study is mostly focused on the intermediate values (Figure 1).
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<p class="texte">The bacterial solutions could not be counted because of two main problems: the too high number of bacteria with the 0.1 OD or the too low number of bacteria with the 0.01 OD. Thus, the study is mostly focused on the intermediate values (Figure 16).
<br/>First of all, a same cell concentration can be noticed with the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio.  
<br/>First of all, a same cell concentration can be noticed with the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio.  
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.
<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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</br>
</br>
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<p class="texte">Figure 1: CBB presence has no effect on bacteria. The bacterial concentration was measured regarding  <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).
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<p class="legend">Figure 16: CBB presence has no effect on bacteria. The bacterial concentration was measured regarding  <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>
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<p class="title3">Results</p>
<p class="title3">Results</p>
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<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration : 105 bacteria/mL (Figure 2). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more Wild Type bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module.   
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<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration : 105 bacteria/mL (Figure 17). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more Wild Type bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module.   
<br/>Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.</p>
<br/>Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.</p>
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</br>
</br>
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<p class="texte">Figure 2:  Attachment of <i>Bacillus subtilis</i> with binding module to chitin. <span style="color:#0000FF">The WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentration has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.</p>
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<p class="legend">Figure 17:  Attachment of <i>Bacillus subtilis</i> with binding module to chitin. <span style="color:#0000FF">The WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentration has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.</p>
<p class="title2">3. Microscopic observations</p>
<p class="title2">3. Microscopic observations</p>
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<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="texte">Figure ***: </p>
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<p class="legend">Figure 18: Microscopic view of bead 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%"><iframe width="420" height="315" src="//www.youtube.com/embed/G3xpYnkUr3o" frameborder="0" allowfullscreen></iframe></center>
<center><img src="http://2014.igem.org/wiki/images/5/53/Photo_billes_microscopie.png" width="45%"><iframe width="420" height="315" src="//www.youtube.com/embed/G3xpYnkUr3o" frameborder="0" allowfullscreen></iframe></center>
</br>
</br>
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<p class="texte">Figure ***: </p>
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<p class="legend">Figure 19: A short movie of 3D bead surfaces coated with chitin</p>
<p class="texte">Finally we want to observe the bacteria after the second wash. When our bacterium has the binding module, results suggest a lower number of bacteria in the washing solution. SubtiTree is retained by the beads.</p>
<p class="texte">Finally we want to observe the bacteria after the second wash. When our bacterium has the binding module, results suggest a lower number of bacteria in the washing solution. SubtiTree is retained by the beads.</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>
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<p class="texte">Figure ***:  
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<p class="legend">Figure 20: Microscopic view of bacteria after washing
</p>
</p>
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</br>
</br>
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<img style="width:400px; " src="http://parts.igem.org/wiki/images/c/c2/Resultfong.jpg"> Figure X
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<img style="width:400px; " src="http://parts.igem.org/wiki/images/c/c2/Resultfong.jpg"> <p class="legend">Figure 21</p>
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<img style="width:400px; " src="http://parts.igem.org/wiki/images/9/92/Results_fong_2.jpg"> Figure X
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<img style="width:400px; " src="http://parts.igem.org/wiki/images/9/92/Results_fong_2.jpg"> <p class="legend">Figure 22</p>
</br>
</br>

Revision as of 16:28, 15 October 2014