Part:BBa_K3463001:Experience

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Characterization of BBa_K3463001

As we didn’t manage to create the PAO1’s biofilm in our laboratory, we sent the Dispersin B BBa_K1659200 to evaluate its activity on P. aeruginosa’s biofilm to Bioaster. For that, their tested conditions were: Lysis buffer (used to obtain the E. coli lysates) Lysate Dispersin B Lysate Pyocin S5 Lysate non-transformed E. coli BL21

These conditions were both inoculated at 25 or 50% in triplicate and compared to a non-traited condition. Then, they counted the CFU/mL per replicate and calculated the bacterial concentrations for each condition. As you can see (Figure 1A), the preliminary data are highly dispersed for the tested conditions. The lysis buffer condition didn’t impact bacterial concentrations and comparable results to the non-treated conditions. Concerning the Dispersin B and the Pyocin S5, they present a dose-effect and the bacterial concentrations are higher than the non-treated condition. It seems that they have a stimulatory effect.

Figure 1 Effect of the therapeutic molecules on the pseudomonas aeruginosa’s biofilm.

The next step would be to reiterate these experiments by optimizing the conditions. To do so, the lysates would be purified to have higher protein concentrations than in the preliminary tests. We could do a western blot with antibodies directed against both Dispersin B and Pyocin S5 to make sure that the proteins are expressed. Moreover, the freezing/thaw process for transport can have an impact on the protein and have to be avoided. Furthermore, the observed effect of our therapeutic molecules didn’t reproduce the one observed in the literature. Therefore further experiments could allow us to fully understand and observe the mechanism of those molecules and to assess their relevance in our therapeutic system.

To evaluate the killing activity of the Pyocin S5 BBa_K3463001 on P. aeruginosa, we performed two different experiments :

The first one (Figure 2) aimed to show the OD decrease of P. aeruginosa’s growing cultures over time incubated with various concentrations of Pyocin S5 and three negative controls.

Figure 2 Protocols experiments used to determine the killing effect of the Pyocin S5 on planktonic pseudomonas aeruginosa. .

The data obtained from this experiment (Figure 3) didn’t show any differences between all the growing cultures after 2H of incubation. Indeed, the OD of P. aeruginosa didn’t decrease or decelerate over the time for cultures with Pyocin S5. Thus, we can say that the Pyocin S5 doesn’t have any killing activity against planktonic P. aeruginosa in suspension at the concentration tested.

Figure 3 Table representing the growth rate of pseudomonas aeruginosa under six different conditions.

The second experiment (Figure 4) showed the same result on an agar plate. We wanted to show if the Pyocin S5 can block the growth of P. aeruginosa on LB agar plates.

Figure 4 Agar plate representing the killing effect of five different samples on pseudomonas aeruginosa

After an overnight culture at 37°C, the plate was analysed and didn’t show any growing inhibition around all the five discs soaked with Pyocin S5. Thus, we can say that the Pyocin S5 doesn’t have any killing or inhibiting activity against P. aeruginosa on LB agar plate.

Nevertheless, these unexpected results can be explained by the sonication protocol (not optimised) and the stockage at -80°C. Indeed, it is possible that these steps led to a loss of the activity of the Pyocin S5 protein.

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