Difference between revisions of "Part:BBa K2978000"

(→‎Usage)
 
(23 intermediate revisions by 3 users not shown)
Line 5: Line 5:
 
This part makes reference to an endolysin from a bacteriophage that infects <i>Clostridium difficile</i>. It is a segment of the complete protein CD27L, which residues from 2 to 143 are homolougous to the domain <i>N-acetyl-muramoyl-L-alanine</i> amidase. This part codes for residues 1 to 179 of the mentioned protein. It has been codon optimized for <i>E.coli</i>. 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 <i>et al.</i>, 2015).
 
This part makes reference to an endolysin from a bacteriophage that infects <i>Clostridium difficile</i>. It is a segment of the complete protein CD27L, which residues from 2 to 143 are homolougous to the domain <i>N-acetyl-muramoyl-L-alanine</i> amidase. This part codes for residues 1 to 179 of the mentioned protein. It has been codon optimized for <i>E.coli</i>. 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 <i>et al.</i>, 2015).
  
−
===Usage===
+
<h2>Usage</h2>
−
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 <i>E. coli</i> 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.
+
The expression of this part is under a T7 promoter, therefore the chasis must contain the T7 polymerase. Considering that this protein is induced with IPTG, this molecule was applied to an <i>E. coli</i> BL21 de3 culture to estimulate lysin production.  
  
−
===Characterization===
+
<h2>Characterization</h2>
−
https://2019.igem.org/wiki/images/e/e0/T--Costa_Rica--team-R1.png
+
For its purification, the lysin was tagged with 6 histidines in the C-terminal and therefore, a Ni-NTA resin column was used. 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).
 +
 
 +
[[File:T--Costa_Rica--team-R1.png|900px|thumb|center]]
 +
 
 +
<p style="font-size: 80%; text-align: center!important">Figure 1: Polyacrylamide gel electrophoresis of
 +
                                endolysin CD27L<SUB>1-179</SUB> purification.</p>
 +
 
 +
<h2>Lysis assay</h2>
 +
The lysis activity of CD27L<SUB>1-179</SUB> was test in <i>Clostridium difficile</i>, <i>Escherichia coli</i> (B), <i>Staphylococcus sp.</i> (C) and <i>Salmonella abatetuba</i> (B). 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;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. Plates were incubated for 48 hours at 37ºC.
 +
No inhibitory halos were observed. Thus, we conclude that this protein CD27L<SUB>1-179</SUB> wasn't able to inhibit their growing.
 +
 
 +
[[File:T--Costa_Rica--team-R2.png|750px|thumb|center]]
 +
<p style="font-size: 80%; text-align: center!important">Figure 2. Lysis assay of endolysin
 +
                                CD27L<SUB>1-179</SUB> on: A) <i>Clostridium difficile</i>, B) <i>Escherichia coli</i>,
 +
                                C) <i>Staphylococcus sp.</i> and D) <i>Salmonella abatetuba</i>. Amoxicillin (AMX, 10 µg) and
 +
                                chloramphenicol (CHL, 30 µg) were tested as positive control agents and PBS as negative control (-); both antibiotics were used by
 +
                                recommendation of Andino-Molina and colleagues (2019). Lysin was assayed with three
 +
                                different concentrations (1: 120 µg/mL, 2: 60 µg/mL and 3: 30 µg/mL) to evaluate its growth
 +
                                inhibition capacity.</p>
  
 
===Sequence and Features===
 
===Sequence and Features===

Latest revision as of 17:12, 13 December 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 protein is induced with IPTG, this molecule was applied to an E. coli BL21 de3 culture to estimulate lysin production.

Characterization

For its purification, the lysin was tagged with 6 histidines in the C-terminal and therefore, a Ni-NTA resin column was used. 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).

T--Costa Rica--team-R1.png

Figure 1: Polyacrylamide gel electrophoresis of endolysin CD27L1-179 purification.

Lysis assay

The lysis activity of CD27L1-179 was test in Clostridium difficile, Escherichia coli (B), Staphylococcus sp. (C) and Salmonella abatetuba (B). 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;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. Plates were incubated for 48 hours at 37ºC. No inhibitory halos were observed. Thus, we conclude that this protein CD27L1-179 wasn't able to inhibit their growing.

T--Costa Rica--team-R2.png

Figure 2. Lysis assay of endolysin CD27L1-179 on: A) Clostridium difficile, B) Escherichia coli, C) Staphylococcus sp. and D) Salmonella abatetuba. Amoxicillin (AMX, 10 µg) and chloramphenicol (CHL, 30 µg) were tested as positive control agents and PBS as negative control (-); both antibiotics were used by recommendation of Andino-Molina and colleagues (2019). Lysin was assayed with three different concentrations (1: 120 µg/mL, 2: 60 µg/mL and 3: 30 µg/mL) to evaluate its growth inhibition capacity.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]