Generator

Part:BBa_K1179071

Designed by: Nelson Hall   Group: iGEM13_MIT   (2013-09-18)

CMV_Acyl-TyA-eGFP

This construct produces the exosome/membrane targeting protein Acyl-TyA-eGFP under the CMV promoter.

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. This image was 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).
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.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2062
    Illegal SpeI site found at 2037
    Illegal PstI site found at 1282
    Illegal PstI site found at 1531
    Illegal PstI site found at 1553
    Illegal PstI site found at 2067
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2062
    Illegal SpeI site found at 2037
    Illegal PstI site found at 1282
    Illegal PstI site found at 1531
    Illegal PstI site found at 1553
    Illegal PstI site found at 2067
    Illegal NotI site found at 2088
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2062
    Illegal BglII site found at 1382
    Illegal BamHI site found at 2031
    Illegal XhoI site found at 2095
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2062
    Illegal SpeI site found at 2037
    Illegal PstI site found at 1282
    Illegal PstI site found at 1531
    Illegal PstI site found at 1553
    Illegal PstI site found at 2067
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2062
    Illegal SpeI site found at 2037
    Illegal PstI site found at 1282
    Illegal PstI site found at 1531
    Illegal PstI site found at 1553
    Illegal PstI site found at 2067
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1215
    Illegal BsaI.rc site found at 1567
    Illegal BsaI.rc site found at 2020


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