Team:Toulouse/Notebook/Protocols

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<br>Three different funguss strains were used : <I>Aspergillus brasiliensis, Aspergillus nidulans, Trichoderma reesei</I>
<br>Three different funguss strains were used : <I>Aspergillus brasiliensis, Aspergillus nidulans, Trichoderma reesei</I>
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.
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<br>- Then the drop is mixed with 1mL of sterile water.
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<br>- Then the drop is mixed with 1mL of sterile water in an Eppedorf.
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<br>- a microscopy count can be performed thanks to Toma cell to determine the conidia concentration.
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<br>- A microscopy count can be performed thanks to Thoma cell to determine the conidia concentration.
<br>NB : The conidia solutions are then diluted and spread on sap medium to get 10,000 conidia/plate.
<br>NB : The conidia solutions are then diluted and spread on sap medium to get 10,000 conidia/plate.
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<br>- After 72hours of liquid culture of different clones of B. subtilis with the fungicides module, the culture can be centrifuged.
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<br>- After 72hours of liquid culture of the different clones of <i>B. subtilis>/i> with the fungicides module, the culture can be centrifugated.
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<br>- 130µL of supernatant is used to soak a plug on a fungus plate. The bacterial pellet is resuspended in 130µL of LB medium and put on a tampon.  
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<br>- 130µL of the supernatant is used to soak a pad placed on the conidia plate. The bacterial pellet is resuspended in 130µL of LB medium and also put on a pad.  
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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.
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<br>- The plates containing 10,000 conidia and the soaked pads are then put at room temperature for a few days according to the growth speed of the fungi. Controls are also realized with wild 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>
<p class="title2">Fungicide test: <i>in planta</i> assay</p>
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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.
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The first step is related to the inoculation of SubtiTree in plants through stomata (opened in wet condition). We diluted our bacterial samples to get two concentrations:  5.10^6 and 10^8 bacteria per mL. The WT and trasnformed bacteria are introduced into plants (a control test without bacteria was performed). Thanks to a 1 ml syringe (without needle), the plant was injected with bacteria by pressure. Five leaves of each plant were used, 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. Bacterial growth in the plant is left for 24 h.
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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.
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The next step begins with the preparation of the fungal samples.Fungal culture is crushed and mixed with PDB (Potato Dextrose Broth). Then the mix passes through a 100 µm filter (to remove large aggregates) and  through a 40 µm filter. The caught hyphae are mixed with PDB for 24 to 48 hours until OD(600nm) 2.5 isobtained. The previously seeded leaves are taken from the plant using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf 5µl of the fungal suspension is placed (using beveled tips because it is too viscous. As control, we kept inoculated leaves without fungus and leaves with only fungus. Pictures are taken at different times. All the plants are destroyed by autoclaving.
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Revision as of 14:59, 16 October 2014