Difference between revisions of "Part:BBa K4645008"
Felicitysm (Talk | contribs) (→Usage and Biology) |
Felicitysm (Talk | contribs) (→Methods and Result) |
||
| Line 10: | Line 10: | ||
===Methods and Result=== | ===Methods and Result=== | ||
| − | <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> | + | <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> |
<br> | <br> | ||
<html> | <html> | ||
| Line 24: | Line 24: | ||
</body> | </body> | ||
<br> | <br> | ||
| − | <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 | + | <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> |
<br> | <br> | ||
<html> | <html> | ||
Revision as of 12:05, 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.
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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 816
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]