Team:Toulouse/Notebook/Protocols

From 2014.igem.org

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<p class="title1" id="select8">Final Tests</p>
<p class="title1" id="select8">Final Tests</p>
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<p class="title2">Chemotaxis test</p>
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<p class="texte">
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<B> Chemotaxis test </B>
 
<br>Many chemotaxis tests exist such as plate tests or capillary tests. We tried all of them and we decided to optimize the « Capillary essay » from Imperial College 2011 iGEM team.
<br>Many chemotaxis tests exist such as plate tests or capillary tests. We tried all of them and we decided to optimize the « Capillary essay » from Imperial College 2011 iGEM team.
<br>- Prepare the bacteria in LB medium so they reach an OD between 0.5 and 0.6 (exponential growth phase). This step takes about 5 hours.
<br>- Prepare the bacteria in LB medium so they reach an OD between 0.5 and 0.6 (exponential growth phase). This step takes about 5 hours.
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<br>- Count colonies on different plates
<br>- Count colonies on different plates
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<p class="title2">Fungicide test: anti-fungal activities</p>
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<p class="texte">
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<B> Fungicide test: anti-fungal activities </B>
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CAUTION : all the lab equipment must be desinfected before the manipulations with the fungi.  
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<br>CAUTION : all the lab equipment must be desinfected before the manipulations with the fungi.  
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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>- 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="title2">Fungicide test: <i>in planta</i> assay</p>
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<p class="texte">
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>
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>

Revision as of 22:50, 15 October 2014