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Team:MIT/GFP
From 2013.igem.org
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| + | <b>Conclusion:</br> | ||
| + | From the following microscopy data, we can conclude that the Acyl-TyA tag is targeting our GFP protein to the membrane and to the site of exosome biogenesis. These results inform later protein fusion experiments, strengthening our confidence that the protein signals fused to Acyl-TyA are making their way into exosomes. | ||
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Revision as of 03:55, 28 September 2013
Overview
To visualize the localization and exosomal exportation of Acyl-TyA, the enhanced green fluorescent protein (eGFP) was added to the C-terminus and the expression of the fusion protein (Acyl-TyA-eGFP) was driven by a human cytomegalovirus (CMV) promoter.
Method:
Previous research on biogenesis of exosomes has shown the rhodamine phosphatidylethanolamine (Rh-PE) preferentially labels the endosome-like-domain (ELD), which is believed to be the exosomal budding site on cell membrane. 24 hours after Jurkat T cells were nucleofected with CMV_Acyl-TyA-eGFP construction, the cells were stained with Rh-PE. The colocalization of the stain and green fluorescence was observed by confocal for 24 hrs.
Results:
Rh-PE was seen to localize at the ELD immediately after staining procedure. Over the course of 24 hrs, exosomes budded out with stain. 48 hrs post nucleofection, the green fluorescence was observed to localize on cell membrane.
Jurkat T cells were transfected with pCMV_Acyl-TyA-GFP and after 24 hours stained with Rh-PE and NucBlue nuclear stain. The following images were taken on the confocal microscope 12 hours later as sequential focal planes. The planes are then stacked together to generate the 3D image.
Here, we overlay both the fluorescence of the green channel and the red channel to show the colocalization of Acyl-TyA-GFP and Rh-PE, indicating that our Acyl-TyA tag is localizing at the ELD (the site of exosome biogenesis).
Conclusion: From the following microscopy data, we can conclude that the Acyl-TyA tag is targeting our GFP protein to the membrane and to the site of exosome biogenesis. These results inform later protein fusion experiments, strengthening our confidence that the protein signals fused to Acyl-TyA are making their way into exosomes.
