Difference between revisions of "Team:Freiburg/Composite Part"
| Line 123: | Line 123: | ||
<td>Composite</td> | <td>Composite</td> | ||
<td>FPR2-receptor with eGFP</td> | <td>FPR2-receptor with eGFP</td> | ||
| − | <td>1791 bp<td> | + | <td>1791 bp</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 129: | Line 129: | ||
<td>Composite</td> | <td>Composite</td> | ||
<td>FPR2-receptor with mCherry</td> | <td>FPR2-receptor with mCherry</td> | ||
| − | <td>1782 bp<td> | + | <td>1782 bp</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 135: | Line 135: | ||
<td>Type</td> | <td>Type</td> | ||
<td>sfGFP with amber inside chrom.</td> | <td>sfGFP with amber inside chrom.</td> | ||
| − | <td>Length<td> | + | <td>Length</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 141: | Line 141: | ||
<td>Type</td> | <td>Type</td> | ||
<td>sfGFP with Amber outside the chrom.</td> | <td>sfGFP with Amber outside the chrom.</td> | ||
| − | <td>Length<td> | + | <td>Length</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 147: | Line 147: | ||
<td>Type</td> | <td>Type</td> | ||
<td>sfGFP with MS2 loops</td> | <td>sfGFP with MS2 loops</td> | ||
| − | <td>Length<td> | + | <td>Length</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 153: | Line 153: | ||
<td>Type</td> | <td>Type</td> | ||
<td>sfGFP (Amber inside chrom.) MS2 loops </td> | <td>sfGFP (Amber inside chrom.) MS2 loops </td> | ||
| − | <td>Length<td> | + | <td>Length</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 159: | Line 159: | ||
<td>Type</td> | <td>Type</td> | ||
<td>sfGFP (Amber outside chrom.) MS2 loops</td> | <td>sfGFP (Amber outside chrom.) MS2 loops</td> | ||
| − | <td>Length<td> | + | <td>Length</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 165: | Line 165: | ||
<td>Type</td> | <td>Type</td> | ||
<td>synthetase with MCP </td> | <td>synthetase with MCP </td> | ||
| − | <td>Length<td> | + | <td>Length</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 171: | Line 171: | ||
<td>Composite</td> | <td>Composite</td> | ||
<td>SPD5 with sfGFP</td> | <td>SPD5 with sfGFP</td> | ||
| − | <td>4320 bp<td> | + | <td>4320 bp</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 177: | Line 177: | ||
<td>Composite</td> | <td>Composite</td> | ||
<td>SPD5 with MCP, synthetase</td> | <td>SPD5 with MCP, synthetase</td> | ||
| − | <td>4890 bp<td> | + | <td>4890 bp</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 183: | Line 183: | ||
<td>Composite</td> | <td>Composite</td> | ||
<td>SPD5 with MCP, sfGFP and synthetase</td> | <td>SPD5 with MCP, sfGFP and synthetase</td> | ||
| − | <td>Length<td> | + | <td>Length</td> |
</tr> | </tr> | ||
Revision as of 12:37, 13 December 2019
Here you can find our composite parts
|
Name
keyboard_arrow_up
keyboard_arrow_down
|
Type
keyboard_arrow_up
keyboard_arrow_down
|
Description
keyboard_arrow_up
keyboard_arrow_down
|
Length
keyboard_arrow_up
keyboard_arrow_down
|
BBa_K3009020 | Composite | FPR2-receptor with eGFP | 1791 bp |
|---|---|---|---|
| BBa_K3009021 | Composite | FPR2-receptor with mCherry | 1782 bp |
| BBa_K3009004 | Type | sfGFP with amber inside chrom. | Length |
| BBa_K3009005 | Type | sfGFP with Amber outside the chrom. | Length |
| BBa_K3009034 | Type | sfGFP with MS2 loops | Length |
| BBa_K3009035 | Type | sfGFP (Amber inside chrom.) MS2 loops | Length |
| BBa_K3009036 | Type | sfGFP (Amber outside chrom.) MS2 loops | Length |
| BBa_K3009037 | Type | synthetase with MCP | Length |
| BBa_K3009038 | Composite | SPD5 with sfGFP | 4320 bp |
| BBa_K3009039 | Composite | SPD5 with MCP, synthetase | 4890 bp |
| BBa_K3009040 | Composite | SPD5 with MCP, sfGFP and synthetase | Length |
This is our favorite composite part: BBa_K3009020
The human formyl peptide receptor 2 (FPR2) is a G-protein coupled receptor which is physiologically expressed on immune cell lineages like neutrophils and T-cells. Among other peptides the FPR2 senses the Staphylococcus aureus toxin PSMα3. It was suggested by Cheung et al 2014 that the binding mechanism relies on the formylated N-terminus of the peptide as well as on the C-terminus. In response to receptor activation FPR2 elicits a signaling cascade depending on calcium ions as second messengers. Ultimately this leads to immune cell activation, secretion of inflammatory cytokines and chemotaxis. All of this is connected with an inflammatory outcome in vivo. The neutrophil activation by FPR2 is therefore an important mechanism of its toxicity because it leads to an aggravation of the inflammation related symptoms in Staphylococcus aureus infection.
This Biobrick can be used in signaling studies or the cellular detection of several small formylated amyloidogenic peptides. As a controls two peptides with inhibitory (WRWW4) and activating (WKYMVm) effect on FPR2 are available.
To investigate the FPR2 receptor we generated a eGFP-tagged FPR2 fusion construct for transfection of HEK293 cells. This construct was designed with an C-terminal HA-tag to be used for antibody staining and GFP for analyzing the cellular localization of FPR2 via microscopy. Transfected HEK293 cells were analyzed by flow cytometry using mouse anti-Human FPR2 coupled to Alexa647 antibody to detect membrane localization of FPR2 (Fig.1)
Knowing that FPR2-eGFP is expressed on the surface of HEK293 cells, we proceeded with calcium influx assays of those cells using our chemically synthesized L-PSMα3. Treating HEK293_FPR2-eGFP cells with 700nM L-PSMα3 an increase in intracellular calcium release was detected (Fig. 2A). Although this was relatively low compared to the ionomycin control, no change in calcium flux was detected in non-transfected HEK293 cells (Fig. 3). The membrane location and specific activation of FPR2 was moreover tested by adding the potent peptide activator WKYMVm (REF) to HEK293_FPR2-eGFP cells (Fig. 2B).
