Difference between revisions of "Part:BBa K3606035"
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<p>The secretion of CaAP(Calcium Absorption Peptide) after its expression is indispensable for it to display the robust function of combining calcium. Here, we decided to achieve CaAP secretion via the type II secretion system, which generally contains two steps. The first step involves some signal peptides targeted to the Sec secretory pathways, which are responsible for transporting CaAP across the inner membrane into the periplasm. For Gram-negative bacteria like E.coli, the second step can then take place once CaAP is in the periplasm, as the intrinsic protein complex GspD creates a pore in the outer cell membrane through which CaAP can be secreted out of the bacteria. </p> | <p>The secretion of CaAP(Calcium Absorption Peptide) after its expression is indispensable for it to display the robust function of combining calcium. Here, we decided to achieve CaAP secretion via the type II secretion system, which generally contains two steps. The first step involves some signal peptides targeted to the Sec secretory pathways, which are responsible for transporting CaAP across the inner membrane into the periplasm. For Gram-negative bacteria like E.coli, the second step can then take place once CaAP is in the periplasm, as the intrinsic protein complex GspD creates a pore in the outer cell membrane through which CaAP can be secreted out of the bacteria. </p> | ||
<p>Part BBa_K3606035 is a new composite part designed to allow CaAP secretion from our engineered E.coli strain by adding a signal peptide alkaline phosphatase (PhoA) to the N terminus of CaAP. As a functional peptide that allows surface anchoring of its fused gene product, PhoA enables CaAP to be presented on cellular surface of E.coli. </p> | <p>Part BBa_K3606035 is a new composite part designed to allow CaAP secretion from our engineered E.coli strain by adding a signal peptide alkaline phosphatase (PhoA) to the N terminus of CaAP. As a functional peptide that allows surface anchoring of its fused gene product, PhoA enables CaAP to be presented on cellular surface of E.coli. </p> | ||
| + | <p>The 5 signal peptides candidates for CaAP secretion are listed below.</p> | ||
| + | <table> | ||
| + | <tr> | ||
| + | <th> Signal Peptide </th> <th> Number for Registry </th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> NSP4 </td> <td> BBa_K3606042 </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> OmpA </td> <td> BBa_K3606043 </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> DsbA </td> <td> BBa_K3606030 </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> PelB </td> <td> BBa_K208004 </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> PhoA </td> <td> BBa_K808028 </td> | ||
| + | </tr> | ||
| + | </table> | ||
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Revision as of 02:19, 28 October 2020
PhoA-CaAP
We fused PhoA to the N-terminal of CaAP for its secretion into the extracellular space.
Usage and Biology
The secretion of CaAP(Calcium Absorption Peptide) after its expression is indispensable for it to display the robust function of combining calcium. Here, we decided to achieve CaAP secretion via the type II secretion system, which generally contains two steps. The first step involves some signal peptides targeted to the Sec secretory pathways, which are responsible for transporting CaAP across the inner membrane into the periplasm. For Gram-negative bacteria like E.coli, the second step can then take place once CaAP is in the periplasm, as the intrinsic protein complex GspD creates a pore in the outer cell membrane through which CaAP can be secreted out of the bacteria.
Part BBa_K3606035 is a new composite part designed to allow CaAP secretion from our engineered E.coli strain by adding a signal peptide alkaline phosphatase (PhoA) to the N terminus of CaAP. As a functional peptide that allows surface anchoring of its fused gene product, PhoA enables CaAP to be presented on cellular surface of E.coli.
The 5 signal peptides candidates for CaAP secretion are listed below.
| Signal Peptide | Number for Registry |
|---|---|
| NSP4 | BBa_K3606042 |
| OmpA | BBa_K3606043 |
| DsbA | BBa_K3606030 |
| PelB | BBa_K208004 |
| PhoA | BBa_K808028 |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 138
- 1000COMPATIBLE WITH RFC[1000]
Characterization
We have successfully cloned NSP4/PhoA/OmpA guided CaAP(Calcium Absorption Peptide) under the regulation of the iPTG inducible promoter, Ptac, and expressed it in E.coli.BL21. As shown in the figure below,PhoA can guide CaAP to secrete into the extracellular space.
