Difference between revisions of "Team:Costa Rica/Results"
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| − | <h2> | + | <h2>Endolysin CD27L<SUB>1-179</SUB></h2> |
| − | <p> | + | <p>CD27L<SUB>1-179</SUB> is the catalytic domain of a bacteriophage endolysin that infects <i>Clostridium difficile</i>, 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). |
| + | 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 <i>E. coli</i> BL21(DE3), nevertheless we couldn’t produce the protein in our delivery system (<i>L. casei</i>) 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. | ||
| + | </p> | ||
| + | |||
| + | <div class="gallery" style="width:70%; margin-left: auto; margin-right: auto;"> | ||
| + | <a target="_blank"> | ||
| + | <img src="https://static.igem.org/mediawiki/2019/e/e0/T--Costa_Rica--team-R1.png" | ||
| + | style="width:100%";class="img-fluid" alt="Responsive image"> | ||
| + | </a> | ||
| + | <div class="desc"> | ||
| + | <p style="font-size:13px!important;">Figure 1.Polyacrylamide gel electrophoresis of endolysin CD27L<SUB>1-179</SUB> purification.</p> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
Revision as of 20:41, 21 October 2019
Here we enlist the requirements met to achieve each possible medal in the competition. We scheduled our activities in order to fulfill every requirement that iGEM established for getting these accolades. 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).
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.
Figure 1.Polyacrylamide gel electrophoresis of endolysin CD27L1-179 purification.
Endolysin CD27L1-179
