Difference between revisions of "Team:Costa Rica/Results"

Revision as of 00:24, 22 October 2019

The results shown here are product of our work in different laboratories of three universities: Costa Rica Institute of Technology (ITCR), National University of Costa Rica (UNA) and the University of Costa Rica (UCR). Each test was made in compliance to the security standards needed to work with each microorganism and reagent.

Endolysin CD27L_1-179

CD27L_1-179 is the catalytic domain of a bacteriophage endolysin that infects Clostridium difficile. It is homologous to the domain of the N-acetyl-muramoyl-L-alanine amidase. This truncation mutation was selected instead of the complete protein because, according to previous studies, it shows a faster lysis to different strains of the pathogen and a high level of selectivity against clostridia compared to the complete protein (Mayer et al., 2011).
We used the T7 promoter for this construct considering its high transcription level and its general inactivation in the absence of IPTG. Also, the protein was tagged with 6 histidines in the C-terminal, to facilitate its purification by affinity chromatography. Is worth to mentioning that, the strain selected is the most common cell strain of this promoter, the E. coli BL21(DE3), nevertheless we couldn’t produce the protein in our delivery system (L. casei) because the sequences never arrived.
The lysin is a soluble intracellular protein, therefore this kind of proteins were the ones used in the Ni-NTA resin purification. Wash 1 had 10 mM Imidazole and Wash 2, 20 mM Imidazole. Then, the protein of interest was eluted in 500 mM Imidazole. As shown in the next SDS-PAGE, considering the size of the band, we confirmed the presence of our purified protein in the elution number 1 (F1).

Figure 1.Polyacrylamide gel electrophoresis of endolysin CD27L_1-179 purification.

Before lysis assays, the protein was dialyzed to remove Imidazole.

Lysis assay

The lysis activity of CD27L_1-179 was tested on Clostridium difficile NCTC 13307, Escherichia coli, Staphylococcus sp. and Salmonella abatetuba. Following the Kirby-Bauer method, bacterias were inoculated in a Mueller-Hinton agar, using different concentration of lysin. Amoxicillin and chloramphenicol were used as positive controls and PBS as negative control. As shown in Figure No inhibitory halos were observed. Thus, we conclude that this protein CD27L1-179 wasn't able to inhibit their growing. The lysis activity of CD27L_1-179 was tested on Clostridium difficile, Escherichia coli, Staphylococcus sp. and Salmonella abatetuba. Following the Kirby-Bauer method, bacterias were inoculated in a Mueller-Hinton agar, using 0.12 mg/mL, 0.06 mg/mL and 0.03 mg/mL of lysin. Amoxicillin and chloramphenicol were used as positive controls and PBS as negative control.
As shown in Figure 2, no inhibitory halos were observed. Thus, we conclude that this protein CD27L_1-179 didn’t show inhibition activity in their growths. Nonetheless, we keep working in the laboratory improving test conditions, as lysin and zinc concentration.

Figure 2. Lysis assay of endolysin CD27L_1-179 on Clostridium difficile (A), Escherichia coli (B), Staphylococcus sp. (C) and Salmonella abatetuba (D). Amoxicillin and chloramphenicol were tested as positive control agent; both antibiotics were used by recommendation of Andino-Molina and colleagues (2019), lysin were essayed with three different concentrations (1: 120µg/mL, 2: 60µg/mL and 3: 30µg/mL) to evaluate its growth inhibition capacity.

class="title-text" id="Lysis assay" References

Andino-Molina, M., Barquero-Calvo, E., Seyboldt, C., Schmoock, G., Neubauer, H., Tzoc, E., Rodríguez, C. & Quesada-Gómez, C. (2019). Multidrug-resistant Clostridium difficile ribotypes 078 and 014/5-FLI01 in piglets from Costa Rica. Anaerobe, 55, 78-82.

Mayer, M. J., Garefalaki, V., Spoerl, R., Narbad, A., & Meijers, R. (2011). Structure-based modification of a Clostridium difficile-targeting endolysin affects activity and host range. Journal of bacteriology, 193(19), 5477–5486. doi:10.1128/JB.00439-11