Difference between revisions of "Part:BBa K2978000"

 
 
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<partinfo>BBa_K2978000 short</partinfo>
 
<partinfo>BBa_K2978000 short</partinfo>
  
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This part makes references to an endolysin from a bacteriophage that infects <i>Clostridium difficile</i>. It is section of the complete protein CD27L, which in the residues 2 to 143 has its domain homologous to <i>N-acetyl-muramoyl-L-alanine</i> amidases. In this part we synthesize  de novo the residues 1 to 179 of the protein, with codon optimization for <i>E.coli</i>. In previously works this reduced protein has shows a faster lysis to different strains of the pathogen and a high level of selectivity against clostridia [1].
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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).
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The expression of this parts is under a T7 promoter, this mean that if we want to induce this protein we have to use a chassis with T7. In our case we confirm that <i>E. coli</i> BL21 de3 produce the lysin when IPTG is applied, because in this strain the polymerase is under a pLac promoter (Figure 1).
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<h2>Usage</h2>
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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.
  
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<h2>Characterization</h2>
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===Usage and Biology===
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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).
  
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[[File:T--Costa_Rica--team-R1.png|900px|thumb|center]]
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2978000 SequenceAndFeatures</partinfo>
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<p style="font-size: 80%; text-align: center!important">Figure 1: Polyacrylamide gel electrophoresis of
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                                endolysin CD27L<SUB>1-179</SUB> purification.</p>
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<h2>Lysis assay</h2>
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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.
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No inhibitory halos were observed. Thus, we conclude that this protein CD27L<SUB>1-179</SUB> wasn't able to inhibit their growing.
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[[File:T--Costa_Rica--team-R2.png|750px|thumb|center]]
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<p style="font-size: 80%; text-align: center!important">Figure 2. Lysis assay of endolysin
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                                CD27L<SUB>1-179</SUB> on: A) <i>Clostridium difficile</i>, B) <i>Escherichia coli</i>,
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                                C) <i>Staphylococcus sp.</i> and D) <i>Salmonella abatetuba</i>. Amoxicillin (AMX, 10 µg) and
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                                chloramphenicol (CHL, 30 µg) were tested as positive control agents and PBS as negative control (-); both antibiotics were used by
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                                recommendation of Andino-Molina and colleagues (2019). Lysin was assayed with three
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                                different concentrations (1: 120 µg/mL, 2: 60 µg/mL and 3: 30 µg/mL) to evaluate its growth
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                                inhibition capacity.</p>
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===Sequence and Features===
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<partinfo>BBa_K2978000 SequenceAndFeatures</partinfo>
  
 
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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]