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

Predation

Our aim is to reinforce the natural predation capacity of B. subtilis and to ensure it is constant. We first assessed that our wild type Bacillus chassis is not able of predation, then we built the operons allowing boosting the predation property.

Preliminary tests:

We tried different testing approaches to evaluate the predatory response of B. subtilis and eventually elaborate a protocol to do the preliminary test. We tested the predation of B. subtilis Wild Type against Pseudomonas fluorescens, a deleterious strain present in the cave. Briefly, the protocol consists in growing both strains in rich medium, mixing them in PBS and monitoring their growth (figure 1). Figure 1: Bacillus subtilis wild type does not feed on Pseudomonas fluorescens. Both strain were grown overnight in rich medium and then mixed in PBS. The growth of both strains were then monitored for 8 hours by plate numeration. The graph represents the ratio between B. subtilis in PBS in presence of P. fluorescens versus B. subtilis alone in PBS (data normalized to time 1H).

We observed no growth benefit when mixing B. subtilis and P. fluorescens versus B. subtilis alone. We conclude that B. subtilis WT predation program is not the strain priority when facing starvation. Other surviving program as competence or sporulation are likely favoured by B. subtilis in such condition. This reinforces the need to prevent these programs by using a spoOA mutant and to promote the predation by overexpressing either the SKF or SDP operons.

SKF

This predation operon is composed of 7 genes for a total of more than 6 Kb. To get rid of restriction sites that could interfere with the cloning steps, we ordered the optimized sequences from IdT as 4 gblocks. From there, our strategy was to do Gibson cloning to obtain the full operon on pSB1C3 in E. coli and then to transfer it in B. subtilis. However, we did not manage to obtain the whole assembly (figure 2), neither partial ones, in spite of about 20 attempts… Figure 2 : Layout of SKF expected biobrick.

SDP

The SDP operon is smaller than the SKF one and it was possible to obtain the optimized sequences as two gblocks. Here again, we were unfortunate and did not get the expected clones in E. coli (figure 3). Figure 3 : Layout of SDP expected biobrick.

To perform trouble shooting, we try an assembly test with just the two gblocks and deposited the product on gel. We observed that the reaction seems to be effective with the presence of a new band corresponding to the combined size of the two gblocks (figure 4). Figure 4: Gibson assemby of the two SDP Gblocks.

Conclusions and perspectives

It seems our Gibson step is fine since we managed to obtain the SDP assembly, but we could not get E. coli transformants when performing the whole experience. The predation system is based on the production of toxins by B. subtilis, and these toxins were reported to be harmful to E. coli (Nandy et al., 2007, FEBS Letters. 581: 151–56). An explanation to our problems could be that SDP and SKF cloning in E. coli results in the bacterium death. We had thought about this problem, but we had believed the expression driven by the pVeg Bacillus promoter to be insufficient for such effect. Perspectives could be to use a tightly regulated promoter to prevent expression during the cloning step in E. coli, or to try a direct transformation of highly competent Bacillus strain.