Difference between revisions of "Part:BBa K3009020"

Revision as of 21:06, 20 October 2019 (view source) Caroruckes (Talk | contribs) ← Older edit		Revision as of 21:18, 20 October 2019 (view source) Caroruckes (Talk | contribs) Newer edit →
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	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.		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.
−	The presence of FPR2 in transfected cells can be checked by flurescence microscopy and FACS. If no anti FPR2 antibody is available a high resolution microscope can even help to determine if FPR2 is located at the membrane, relying on the GFP reporter.
		+	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)
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	<figure>		<figure>
	<img style="width:50%;"src="https://2019.igem.org/wiki/images/e/e9/T--Freiburg--HEK_FPR2-eGFP_transfection.png">		<img style="width:50%;"src="https://2019.igem.org/wiki/images/e/e9/T--Freiburg--HEK_FPR2-eGFP_transfection.png">
−	<figcaption> <b>Fig.1:</b>  Transfection of HEK cells with FPR2-eGFP in different concentrations. Mouse anti-Human FPR2, Alexa647 is used for detection in flow cytometry. us=unstained ut=untransfected	+	<figcaption> <b>Fig. 1:</b>  Transfection of HEK239 cells with eGFP-tagged FPR2 fusion construct in different concentrations. Mouse anti-Human FPR2, Alexa647 is used for detection in flow cytometry. us=unstained ut=untransfected
	</figure>		</figure>
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		+	Knowing that FPR2-GFP 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-GFP 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.
		+	The membrane location and specific activation of FPR2 was moreover tested by adding the potent peptide activator WKYMVm (REF) to HEK293_FPR2-GFP cells (Fig. 2B).
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		+
	<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 activator WKYMVm (B) leads to calcium flux	+	<figcaption> <b>Fig. 2:</b> Calcium release of transfected FPR2-eGFP HEK cells. A) PSMa3 toxin and activator WKYMVm (B) lead to calcium release
	</figure>		</figure>
	</html>		</html>

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Revision as of 21:18, 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)

Knowing that FPR2-GFP 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-GFP 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. The membrane location and specific activation of FPR2 was moreover tested by adding the potent peptide activator WKYMVm (REF) to HEK293_FPR2-GFP cells (Fig. 2B).

Sequence and Features