Difference between revisions of "Part:BBa K2978000"

Revision as of 03:26, 21 October 2019

Causes cell lysis of the pathogen Clostridium difficile

This part makes reference to an endolysin from a bacteriophage that infects Clostridium difficile. It is a segment of the complete protein CD27L, which residues from 2 to 143 are homolougous to the domain N-acetyl-muramoyl-L-alanine amidase. This part codes for residues 1 to 179 of the mentioned protein. It has been codon optimized for E.coli. In previous works this domain has shown a faster lysis to different strains of the pathogen and a high level of selectivity against clostridia compared to the complete protein (Wang et al., 2015).

Usage

The expression of this part is under a T7 promoter, therefore the chasis must contain the T7 polymerase. Considering that this promoter is induce with IPTG, this molecule was applied to an E. coli BL21 de3 culture to estimulated lysin production. For its purification, the lysin was tagged with 6 histidines in the C-terminal, using a Ni-NTA resin column. Protein was washed with 10 and 20 mM Imidazol and then, eluted in 500 mM Imidazole. After dialyzation, the protein was stored in 50 mM Tris Buffer pH 7.45.

Characterization

Usage

The expression of this part is under a T7 promoter, therefore the chasis must contain the T7 polymerase. Considering that this promoter is induce with IPTG, this molecule was applied to the E. coli BL21 de3 culture to estimulated lysin production. For its purification, the lysin was tagged with 6 histidines in the C-terminal, using a Ni-NTA resin column. Protein was washed with 10 and 20 mM Imidazol and then, eluted in 500 mM Imidazole. After dialyzation, protein was stored in 50 mM Tris Buffer pH 7.45.

Sequence and Features