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ClyR is a chimeric lysin, comprising a cysteine, histidine-dependent amidohydrolase/peptidase catalytic domain from PlyC lysin, and the cell-wall binding domain from PlySs2 lysin. The signal peptide OmpA enables ClyR to be secreted into the periplasmic space of bacteria.
 
ClyR is a chimeric lysin, comprising a cysteine, histidine-dependent amidohydrolase/peptidase catalytic domain from PlyC lysin, and the cell-wall binding domain from PlySs2 lysin. The signal peptide OmpA enables ClyR to be secreted into the periplasmic space of bacteria.
  
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===Sequence and Features===
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<partinfo>BBa_K4645008 SequenceAndFeatures</partinfo>
  
  
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===Result===
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===Design and Expectation===
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<p>We hoped that ClyR could be secreted into the extracellular space to achieve its bactericidal effect, so OmpA was selected as the signal peptide.</p>
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===Methods and Result===
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<p>We used homologous recombination in which the OmpA signal peptide was added to the N-terminus of ClyR and transformed into <i>E. coli</i> DH5α competent cells and incubated overnight at 37℃. Below is a plot of colony PCR results.</p>
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<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/clyr/junp.png" style="width:60%; "></center>
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<center><b>  Figure 1.</b> Colony PCR assay of the OmpA-ClyR fragment. </center>
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<p><i> E. coli</i> strain was cultured to an optical density (OD<sub>600</sub>) of ~0.6, induced by adding IPTG to 0.2 mM, and allowed to grow overnight at 16℃. Purification was performed following the instructions of Ni<sup>2+</sup>-affinity chromatography. Below are the result of protein purification.</p>
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<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/clyr/ompa-clyr-p.png" style="width:40%; "></center>
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<center><b>Figure 1.</b> Colony PCR assay of the OmpA-ClyR fragment.  </center>
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<center><b>Figure 2.</b> SDS-PAGE analysis of OmpA-ClyR </center>
 
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<p>We used homologous recombination in which the OmpA signal peptide was added to the N-terminus of ClyR and transformed into <i>E.coli</i> DH5α competent cells and incubated overnight at 37℃. Below is a plot of colony PCR results.</p>
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Latest revision as of 12:32, 12 October 2023

A chimeric lysozyme with secretory ability.

ClyR is a chimeric lysin, comprising a cysteine, histidine-dependent amidohydrolase/peptidase catalytic domain from PlyC lysin, and the cell-wall binding domain from PlySs2 lysin. The signal peptide OmpA enables ClyR to be secreted into the periplasmic space of bacteria.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 816
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 184
    Illegal AgeI site found at 268
    Illegal AgeI site found at 583
    Illegal AgeI site found at 766
  • 1000
    COMPATIBLE WITH RFC[1000]


Design and Expectation

We hoped that ClyR could be secreted into the extracellular space to achieve its bactericidal effect, so OmpA was selected as the signal peptide.

Methods and Result

We used homologous recombination in which the OmpA signal peptide was added to the N-terminus of ClyR and transformed into E. coli DH5α competent cells and incubated overnight at 37℃. Below is a plot of colony PCR results.


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Figure 1. Colony PCR assay of the OmpA-ClyR fragment.


E. coli strain was cultured to an optical density (OD600) of ~0.6, induced by adding IPTG to 0.2 mM, and allowed to grow overnight at 16℃. Purification was performed following the instructions of Ni2+-affinity chromatography. Below are the result of protein purification.


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Figure 2. SDS-PAGE analysis of OmpA-ClyR