Team:Toulouse/Result/experimental-results

From 2014.igem.org

(Difference between revisions)
Line 129: Line 129:
       <div class="centering" style="padding-top: 20px; padding-bottom:40px;">
       <div class="centering" style="padding-top: 20px; padding-bottom:40px;">
-
<p class="texte">How did we confirm our three different modules and how did we improve our new test? Click on these next titles to see SubtiTree abilities.</p>
+
<p class="texte">What were the results of our experiments ? Click on these next titles to see SubtiTree abilities.</p>
<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a>  | <a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a></p>
<p style="font-size:1.3em; margin: 0 0 30px 0;"><a href="#" onClick="ddaccordion.collapseall('technology'); return false">Collapse all</a>  | <a href="#" onClick="ddaccordion.expandall('technology'); return false">Expand all</a></p>
Line 137: Line 137:
-
<p class="texte">For this module, we performed several tests to prove the existence of chemotaxis in <i>Bacillus subtilis</i> wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine.
+
<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.  
</p>
</p>
 +
<p class="title2">1. Petri Dishes Test </p>
-
<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>.
+
<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>
-
The first one was a protocol from <a href="http://2011.igem.org/Team:Imperial_College_London/Protocols_Chemotaxis">the Imperial College 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>
-
<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>
+
<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>
-
<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>
+
<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>
Line 152: Line 152:
<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="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. 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>
-
Anyway we did not have enough reproducible and reliable results to be satisfied with this test. Furthermore if we are forced to add LB to sugar to observe something, it is hard to distinguish between attracting and chemotaxis effects.</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>
We have started new tries using different protocols</p>
We have started new tries using different protocols</p>
Line 160: Line 160:
<p class="texte">
<p class="texte">
-
This protocol on which we worked is taken from a thesis (ref thèse). <i>B.subtilis</i> are grown overnight and if necessary bacteria cells are concentrated by centrifugation. 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 tetracycline at 25µg/mL thus growth are stopped. Plate are cooled and dried, 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 analyzed results.
+
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.
</p>
</p>
Line 167: Line 167:
<p class="texte">
<p class="texte">
-
On our first try with <i>B. subtilis</i>, 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 tetracycline.
+
On our first try with <i>B. subtilis</i>, we made three wells per plate (Figure 4).The wells were filled with glucose at different concentrations and tetracycline was not added in one of the plates.
</p>
</p>
Line 174: Line 174:
<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 tetracycline. Tetracycline concentration seems to be too large and inhibit our bacteria. Thereafter we have work with tetracycline at 15µg/mL.
+
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.
-
We retry this protocol with less tetracycline. 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.
+
We retry this protocol with this new condition. We made two wells per plate (Figure 5), one with either Glucose or n-acetyl-glucosamide and one with LB medium. As previsously, no results were achieved after 1h, but after 12h we could notice halos.
</p>
</p>
Line 182: Line 182:
<p class="texte">
<p class="texte">
-
Results are not as clear as the first time, but we observe halo around well with glucose at 250mM with and without tetracycline. We have made tries with N-acetyl-glucosamide and we see no halo, this show that our strain <i>B. subtilis</i> 168 is not attracted by N-acetyl-glucosamide.</br>
+
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.</br>
-
Results are not enough clear and reliable with plug-in-pond. We do not understand why we have to wait 12 hours to see halos. So we tried other protocols.
+
However, the results are not enough clear, reliable and reproducible with the plug-in-pond protocol. Another testing protocol was then adopted.  
 +
 
</p>
</p>

Revision as of 14:19, 16 October 2014