Team:Toulouse/Result/experimental-results

From 2014.igem.org

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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>
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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>
<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 16).
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<p class="texte">The bacterial solutions could not be counted because of two major 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).
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<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.  
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<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 agrees 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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<p class="title3">Purpose</p>
<p class="title3">Purpose</p>
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<p class="texte">We want to observe the SubtiTree's binding on beads coated with chitin. In order to perform a 3D reconstruction showing this interaction, we use confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we can highlight the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determine the minimum threshold to remove the background noise and the natural fluorescence.</p>
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<p class="texte">We want to observe the SubtiTree's binding on beads coated with chitin. In order to perform a 3D reconstruction showing this interaction, we use confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we can highlight the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determined the minimum threshold to remove the background noise and the natural fluorescence.</p>
<p class="title3">Results</p>
<p class="title3">Results</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>
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<p class="legend">Figure 18: Microscopic view of bead surfaces coated with chitin</p>
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<p class="legend">Figure 18: Microscopic view of beads 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>
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As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br>
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br>
After several days at 30°C, the PDA (Potato Dextrose Agar) plates covered with fungus and commercial peptides were analyzed.</p></p>
After several days at 30°C, the PDA (Potato Dextrose Agar) plates covered with fungus and commercial peptides were analyzed.</p></p>
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<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions.As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below.  This corresponds to a sporing halo in response to the stress generated by the fungicide.
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<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions. As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below.  This corresponds to a sporulating halo in response to the stress generated by the fungicide.
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<p class="texte">Given these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.
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<p class="texte">Regarding these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.
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<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br>
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br>
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The transformed <i>Bacillus subtilis</i> strains grew at 37°C during 72h and were tested. 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>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the operon GAFP-1 + D4E1 (see the photos below).</br>
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The transformed <i>Bacillus subtilis</i> strains grew at 37°C during 72h and were tested. 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>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the GAFP-1 + D4E1 operon (see the photos below).</br>
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br>
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br>
Regarding EcAMP and the triple-fungicides operon, 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 and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.
Regarding EcAMP and the triple-fungicides operon, 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 and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium.  
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium.  
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Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliansis</i>. This effect is comparable to the one previously noted with low concentration of sulfate copper. </br>
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Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliansis</i>. This effect is comparable to the one previously noted with a low concentration of sulfate copper. </br>
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<p class="texte">
<p class="texte">
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The goal of the project is to introduce the trasnformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br>
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The goal of the project is to introduce the transformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br>
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves.  </br>
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves.  </br>
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br>
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br>
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<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants don't have deleterious effects.</br>
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<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants do not have deleterious effects.</br>
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Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem reduced by <i>B. subtilis</i>  expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br></br>
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Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem to be reduced by <i>B. subtilis</i>  expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br></br>
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br>
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br>
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We can therefore conclude that when SubtiTree is in plant physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves' necrosis </b>.
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We can therefore conclude that when SubtiTree is in plant's physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves necrosis. </b>
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Revision as of 19:41, 16 October 2014