Difference between revisions of "Team:Toulouse France/Experiments"

The predation module was composed of genes from B. subtilis, so we expected it to be expressed only in these bacteria. We designed the operons and made it synthetize. Considering their sizes, we could not obtain them at once and we had to divide them in blocks of two kilobase pairs. From there, our strategy was to do Gibson cloning to obtain the full operons in E. coli and then transfer them in B. subtilis. We managed to obtain the sdp operon but could not transform bacteria with it, probably because it produces a toxin active against E. coli. With more time, we should have been capable of integrate it in a Bacillus plasmid.

Photo du gel

We elaborate a protocol and did the preliminary tests needed to evaluate the predatory response of B. subtilis. We tested the predation of B. subtilis Wild Type against Pseudomonas fluorescens with three different approaches.
First, we aimed to demonstrate B. subtilis was able feed on P. fluorescens and grow with it as sole source of nutrients. Therefore, after an overnight growth of both strains in rich medium, we put them in co-culture in minimal medium and monitored their growth rate all along the day. We saw no growth when doing it with B. subtilis WT.

Then, we did a test on solid medium using cellulose disks soaked in a B. subtilis culture. We displayed them on plates containing a minimal medium seeded with P. fluorescens. If B. subtilis were capable of predation, it would have grown with the nutrients furnished by P. fluorescens lysis, but we observed nothing like that with B. subtilis WT.
Finally, we wanted to test the efficiency of the lysis proteins produced by the bacteria. To do so, we put B. subtilis WT in rich medium to let it express the killing factor and delay protein. Then, we discarded the cells themselves thanks to a centrifugation and put this supernatant in contact with P. fluorescens for twenty minutes. At last, we spread the Pseudomonas on plates of rich medium and let them grow. We compared the growth of cells in contact with the B. subtilis supernatant with the one of cells that did not go through any treatment. With B. subtilis WT, we saw no differences.

Demonstrate :

Since its opening the Lascaux cave frescos are under different fungi’s threat. Conventional treatment have already been tested over the years, they did not work in the long term. It lets the microbiom empty which stimulated the development of other contaminations as fungi.

For this competition we decided to develop an experimental fungal control with a bacteria Bacillus subtilis. This strain is able to produce anti fungal compounds in response to N-acetyl-glucosamin, an important fungi cell wall compound. Our purpose is to find a way to save the Lascaux cave frescos with an innovating treatment which permit to let the microbiom intact and stop the fungal dispersion on the fresco painting.

The first part of our work was to determine the best culture conditions for each tested organisms including our bacteria and all of the fungi strains. We found that the best culture medium containing with ¼ PDA and 2% Glucose. This medium did not permit an optimal bacterial growth but we decided to accept this limit knowing that it will be too much work for such a limited time. Knowing this limit, it is important to note that our results could not be as optimal as expected.

We transformed Bacillus subtilis with two construction : Antifungal A and Antifungal B. They are both large spector antifungal. Our constructions contain the D4E1 and GAFP1 genes which were used by the IGEM team 2014. We decided to use their results as proof of their efficiency. The other antifungal gene are the Dermaseptin b1 gene and the Metchikowin gene (cut or not).

We tested our transformed bacteria and their secretome on three different fungi : Aspergillus niger, Talaromyces funiculosus and Chaetomium globosum.

The main results were obtained on Talaromyces funiculosus. We observed that with our construction antifungal A an inhibition halo around the patch for the supernatant. These observations allow us to conclude that D4E1 has an effect on fungi and a possible cut metchikowin antifungal effect but we need to try them independently to confirm there antifungal actions. To notice, Metchikowin have also an antibacterial function and might disturb the gram positive bacteria’s growth and the antifungal peptides expression.

We did not success to clone our antifungal B construction. We could not test its effect on our fungi target.

Test of the N-acetyl-glucosamine answer :

In order to express specifically the antifungal peptides, we choose two N-acetyl-glucosamine sensitive promotors. We tested their activity with a RFP gene in presence of glucose or N-acetyl-glucosamine. We observed a late and unspecific RFP expression. These promotors need to be optimized. image de test Pnag

Test on rock :

Even if our project could not be used on the Lascaux cave fresco for now, we decided to test the development of our transformed bacteria on piece of stone covered of painting like the Lascaux cave’s one. We do not have the results for the moment. image des tests sur pierre.