Difference between revisions of "Part:BBa K3009020"
Caroruckes (Talk | contribs) |
Caroruckes (Talk | contribs) |
||
Line 26: | Line 26: | ||
<figure> | <figure> | ||
<img style="width:50%;"src="https://2019.igem.org/wiki/images/b/bf/T--Freiburg--FPR2_calcium.png"> | <img style="width:50%;"src="https://2019.igem.org/wiki/images/b/bf/T--Freiburg--FPR2_calcium.png"> | ||
− | <figcaption> <b>Fig. 2:</b> Calcium release of transfected FPR2-eGFP HEK cells. A) PSMa3 toxin and | + | <figcaption> <b>Fig. 2:</b> Calcium release of transfected FPR2-eGFP HEK cells. A) PSMa3 toxin and B) activator WKYMVm lead to calcium release |
</figure> | </figure> | ||
</html> | </html> | ||
+ | |||
+ | |||
+ | <html> | ||
+ | <figure> | ||
+ | <img style="width:50%;"src="https://2019.igem.org/wiki/images/d/da/T--Freiburg--neg_ctrl_calcium.png"> | ||
+ | <figcaption> <b>Fig. 1:</b> Calcium release of non transfected Jurkat cellline A) PSMa3 toxin and B) activator WKYMVm lead to no calcium release | ||
+ | </figure> | ||
+ | |||
+ | |||
+ | |||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here |
Revision as of 22:31, 20 October 2019
FPR2-receptor with eGFP
The human formyl peptide receptor 2 (FPR2) is a G-protein coupeled receptor which is physiologically expressed on immune cell lineages like neutrophils and T-cells. Amongst other peptides the FPR2 senses the staphylococcus aureus toxin PSMa3. 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 signalling 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 signalling studies or the cellular detection of several small formylated amyloidogenic peptides. As a controls two peptides with inhibitry (WRWW4) and activating (WKYMVm) effect on FPR2 are availabe.
To investigate the FPR2 receptor we generated a ePFP-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, Alexa647 antibody to detect membrane localization of FPR2 (Fig.1)