Difference between revisions of "Team:Costa Rica/Results"
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| − | <p>The results shown here are product of our work in different laboratories | + | <p>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 | 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 | Costa Rica (UCR). Each test was made in compliance to the security standards needed to work with | ||
| − | each microorganism and | + | each microorganism and reagent.</p> |
</div> | </div> | ||
Revision as of 00:12, 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. CD27L1-179 is the catalytic domain of a bacteriophage endolysin that infects
Clostridium difficile, homologous to the domain of the N-acetyl-muramoyl-L-alanine
amidase. This truncation mutant was selected instead of the complete protein because it showed a
faster lysis to different strains of the pathogen and a high level of selectivity against
clostridia compared to the complete protein in previous works (Mayer et al., 2011).
Figure 1.Polyacrylamide gel electrophoresis of
endolysin CD27L1-179 purification. Before lysis assays, the protein was dialyzed to remove Imidazole. The lysis activity of CD27L1-179 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 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 CD27L1-179 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 different
concentration of lysin. Amoxicillin and chloramphenicol were used as positive controls and PBS
as negative control.
Figure 2. Lysis assay of endolysin
CD27L1-179 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. 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
Endolysin CD27L1-179
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. 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.
Lysis assay
As shown in Figure 2, no inhibitory halos were observed. Thus, we conclude that this protein
CD27L1-179 didn’t show inhibition activity in their growths. Nonetheless, we keep
working in the laboratory improving test conditions. 
References