Team:Toulouse/Notebook/Protocols

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<br>- The plates containing 10,000 conidia and the supernatant or bacteria soaked plugs are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wilt type strains or copper sulfate at 10 and 20mg/mL.
<br>- The plates containing 10,000 conidia and the supernatant or bacteria soaked plugs are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wilt type strains or copper sulfate at 10 and 20mg/mL.
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<p class="title2">Fungicide test: <i>in planta</i> assay </p>
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<p class="texte">
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<B> Fungicide test: <i>in planta</i> assay </B>
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The first step involves doing  the inoculation of SubtiTree in plants through stomata (opened in wet condition). We dilute our bacterial samples for two concentrations:  5.10^6 and 10^8 bacteria per mL. The bacterial solutions of WT, having the expression cassette of the D4E1 or D4E1-operon GAFP1  are introduced into plants (a control test without bacteria was performed). Thanks to a 1 ml syringe (without needle),we  inject plant with bacteria by pressure. We use five leaves of each plant, marked with a marker point. It is necessary to repeat the operation on both sides of the leaf and the excess is wipe off. The plants are placed in a growth chamber (phytotron) to control light, high humidity, temperature and bacterial non-proliferation. Compared for Agrobacterium species, bacterial growth in the plant is left for 24 h.</br>
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<br> The first step involves doing  the inoculation of SubtiTree in plants through stomata (opened in wet condition). We dilute our bacterial samples for two concentrations:  5.10^6 and 10^8 bacteria per mL. The bacterial solutions of WT, having the expression cassette of the D4E1 or D4E1-operon GAFP1  are introduced into plants (a control test without bacteria was performed). Thanks to a 1 ml syringe (without needle),we  inject plant with bacteria by pressure. We use five leaves of each plant, marked with a marker point. It is necessary to repeat the operation on both sides of the leaf and the excess is wipe off. The plants are placed in a growth chamber (phytotron) to control light, high humidity, temperature and bacterial non-proliferation. Compared for Agrobacterium species, bacterial growth in the plant is left for 24 h.</br>
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<br>The next step begins with preparation of the fungal samples.PDA cores containing the fungal culture is crushed and left in culture in the PDB . Then it pass through a filter of 100 µm (to remove large aggregates) and  through a 40 µm filter to allow small molecules. The caught hyphae are placed in the PDB for 24 to 48 hours. This liquid culture is filtered to retain the living fragment and place in solution. Then, 2.5 of OD is adjusted. Obtained leaves above are detached from the plants using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf is deposited 5μL of fungal solution (using beveled tips because it is too viscous), as control we keep inoculated leaf without fungus. Pictures are achieved at different times. All the plants are destroyed by autoclaving.
<br>The next step begins with preparation of the fungal samples.PDA cores containing the fungal culture is crushed and left in culture in the PDB . Then it pass through a filter of 100 µm (to remove large aggregates) and  through a 40 µm filter to allow small molecules. The caught hyphae are placed in the PDB for 24 to 48 hours. This liquid culture is filtered to retain the living fragment and place in solution. Then, 2.5 of OD is adjusted. Obtained leaves above are detached from the plants using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf is deposited 5μL of fungal solution (using beveled tips because it is too viscous), as control we keep inoculated leaf without fungus. Pictures are achieved at different times. All the plants are destroyed by autoclaving.

Revision as of 22:48, 15 October 2014