Team:Toulouse/Result/experimental-results

The first experiment deals with the culture conditions to see if Bacillus subtilis can resist to a low temperature and with the CBB buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different Bacillus subtilis solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell was performed.

Results

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 focudes on the intermediate values (Figure 1).
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. Morever, 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.
Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.
FIGURE A METTRE

Figure 1 : CBB presence has no effect on bacterial. The bacterial concentration was measured regarding the presence () or the absence of CBB () for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).

Transformed Bacillus subtilis with the binding module is able to produce a protein composed of the bacterial peptidoglycan bonding of LycT and the GbpA 4th domain of Vibrio cholerae allowing the chitin bonding. The synthetic bacterium is put with special beads composed of the polymer miming the fungal pathogen wall. After several washes, bacteria specificaly attached to the chitin are put on plates and counted.

Results

The first observation is that both bacterial solutions of wild type Bacillus subtilis and SubtiTree have the same concentration : 105 bacteria/mL (Figure 2). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more WT bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module.
Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.
FIGURE A METTRE !

Figure 2 : Attachment of Bacillus subtilis with binding module to chitin. The WT bacteria concentration () or the bacteria with the binding system () has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p < 0.05.

Tests with commercial peptides

Results
The first tests were performed with commercial GAFP-1 and D4E1 with different concentrations (12,5µM 25µM 100µM). These tests were performed on different fungal strains sharing the same phylum with Ceratocystis Platani. As Ceratocystis Platani is pathogenic, we couldn't perform tests directly with this fungus.
After several days at 30°C, the PDA (Potato Dextrose Agar) plates covered with fungus and commercial peptides were analyzed. An inhibiton halo was noticeable with commercial D4E1 at 100µM on aspergillus brasiliensis. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, no effect was noticeable in the tested conditions. As positive control, a well-known chemical fungicide was used : 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. This corresponds to a sporing halo in response to the stress generated by the fungicide.
Au vu de ces premiers résultats, nous avons remarqué que de fortes concentrations en antifongiques sont nécessaires pour inhiber la croissance du champignon. Suite à ces tests, de nouvelles conditions ont été adoptées afin de ne pas trop favoriser la croissance du champignon au détriment de la croissance bactérienne. Le milieu de croissance élaboré imite la sève et les incubations ont lieu à température ambiante afin d’être plus proche des conditions trouvées dans l’arbre.

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