Team:Toulouse/Result/experimental-results

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<B>Results</B>
<B>Results</B>
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     The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12,5µM/25µM/100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani<i/>. <br>As <i>Ceratocystis Platani<i/> is pathogenic, we could not perform tests directly with this fungus. <br/>
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     The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12,5µM/25µM/100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>. <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.
After several days at 30°C, the PDA (Potato Dextrose Agar) plates covered with fungus and commercial peptides were analyzed.
<br> FIGURE <br/>
<br> FIGURE <br/>
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     An inhibiton 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.  
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     An inhibiton 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.  
<br>    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.  
<br>    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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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.  
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.  
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<br><br/>The transformed <i>Bacillus subtilis<i/> strains were grown at 37°C during 72h and tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads disposed 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 <br><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.  
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<br><br/>The transformed <i>Bacillus subtilis</i> strains were grown at 37°C during 72h and tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads disposed 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 <br><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.  
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.  
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.  
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 modification 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 modification 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.  
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The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis<i/> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis<i/> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.  
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The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.  
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Revision as of 09:40, 7 October 2014