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From 2013.igem.org

Overview

The highly oligomeric cytoplasmic protein TyA with the N-terminal acylation tag (MGCINSKRKD-) has been shown to be targeted to exosomal budding sites on the plasma membrane and to be sorted into exosomes. When eGFP is added to the fusion of the acylation tag and TyA (Acyl-TyA), the packaging effect was visually observed under microscope. The Acyl-TyA protein is used to transport the desired reverse tetracycline-controlled transactivator (rtTA) to receiver cells through exosomes.

To test the function of rtTA in single cells, a constitutive expression of rtTA is driven by the human elongation factor 1a (hEF1a) promoter. An inducible expression of enhanced blue fluorescent protein (eBFP) is obtained when the eBFP is fused with tetracycline response element (Tre) promoter. Jurkat T cells were nucleofected with both plasmids and incubated with doxycycline (Dox) concentration?. The blue fluorescence was observed 48 hours post nucleofection.

To test the plasma membrane localization effect of Acyl-TyA on rtTA, rtTA is added to the Acyl-TyA sequence with 3 glysines as a linker in between. Both rtTA and Acyl-TyA-rtTA are constitutively expressed. The inducible fluorescence of eBFP as described above was measured by fluorescence activated cell sorter (FACS) machine. The cell count of blue florescence when only rtTA was present was significantly higher than the one when rtTA was fused with Acyl-TyA, which suggested the amount of Acyl-TyA-rtTA present in the cytoplasm was lower than the amount of rtTA. To further test the exportation of Acyl-TyA-rtTA into exosomes, a histin tag is added to the C terminus. After extracted and purified, exosomes secreted from cells that express Acyl-TyA-rtTA were western blotted against anti-his antibody. Do we have result for this? I remembered someone told me the anti his antibody might be defective.

Circuit One:

Circuit Two:

Exosome isolated circuit:

Jurkat isolated circuit: