Team:Toulouse/Result/experimental-results

From 2014.igem.org

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This protocol was adapted 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. 10 mL of the culture was mixed with 15 mL 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.
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This protocol was adapted 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. 10 mL of the culture was mixed with 15 mL 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.
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- The same process was made with xylose as a positive control.<br>
- The same process was made with xylose as a positive control.<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>
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<i>NB: According to the paper "Chemotaxis towards sugars by </i>B. 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>
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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>
- 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>
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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>
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>
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At the beginning, the experiment was conducted with only one negative control, the fuchsin and different NAG concentrations: 25 mM, 250 mM and 500 mM. 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: 25 mM, 250 mM and 500 mM. The tested strain was <i>B. subtilis</i> 168:<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>
<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 <i>Bacillus subtilis</i> was indeed naturally attracted to NAG.</p>
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<p class="texte"> The miracle arrived! We managed to prove that our WT <i>B. subtilis</i> 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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<p class="title2">2. Test with antifungal bacteria</p>
<p class="title2">2. Test with antifungal bacteria</p>
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<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>
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<p class="texte">In order to test our <i>B. 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>
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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>
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The transformed <i>B. 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, we thus suppose a putative 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-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.
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.

Revision as of 03:04, 18 October 2014