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 observed under a microscope. The Acyl-TyA protein is used to transport the desired reverse tetracycline-controlled transactivator (rtTA3, here referred to as rtTA) to receiver cells through exosomes.

To test the functionality of rtTA in single cells, a constitutive expression of rtTA was driven by the human elongation factor 1a (hEF1a) promoter. A receiver circuit consisting of mKate fused to tetracycline response element (Tre) promoter was also constructed. In theory, the constitutive rtTA would drive the expression of mKate in the presence of doxycycline (DOX). Jurkat T cells were nucleofected with both plasmids and incubated with doxycycline (DOX). The mKate fluorescence was observed 48 hours post nucleofection, proving that the rtTA worked.

To test the plasma membrane localization effect of Acyl-TyA on rtTA, rtTA was added to the Acyl-TyA sequence. Due to concerns about the oligemerization effects of TyA hampering the functionality of rtTA, we added a "linker" sequence (three glycines) between TyA and rtTA to space the two proteins out. Later on, once this construct was proved to work, we appended a tag consisting of six histines to the end of the rtTA.

Characterization

Since it is such a crucial component in many of our exosomal cargoes, rtTA needed to be tested extensively before we could attempt to send it. In our project, rtTA is used in conjunction with doxycycline (DOX) and the TRE-tight promoter to activate genes—when DOX is absent, rtTA inhibits the promoter; conversely, when DOX is present, rtTA activates the promoter, allowing transcription of our desired protein. In this case, the output of each circuit is a fluorescent protein.

Also, as it is part of larger fusion proteins, we needed to verify its functionality. Before our verification experiments it was unclear whether the Acyl-TyA section would interfere with rtTA.

First, we needed to ensure that our rtTA sequence was correct and functional. We transfected HEK-293 cells with three circuits: constitutively expressed rtTA, mKate under the influence of the Tre-tight promoter, and constitutively expressed eBFP (used as a transfection marker only). We expected to see red fluorescence when doxycycline was added, and this was indeed the case.

Once we determined that rtTA was functional, we checked the functionality of the Acyl-TyA-rtTA fusion protein. There were concerns that the oligemerization effects of TyA would interfere with the activity of rtTA. We transfected the new Acyl-TyA-rtTA construct into HEK-293 cells, using mKate for the output and eBFP for the transfection marker like in the previous experiment. We found that the fusion rtTA still worked in the presence of doxycycline.

As previously stated, both rtTA and Acyl-TyA-rtTA worked as we had hoped. The fusion protein had somewhat reduced effectiveness, most likely due to hindrance from TyA; however, its activity was sufficient for our purposes.

The above graph shows the red fluorescence detected for Acyl-TyA-rtTA and the lone rtTA both with and without DOX. With DOX, both rtTA circuits activated the mKate. As mentioned above, rtTA alone (the green line) was more effective, and despite its lower activity, Acyl-TyA-rtTA (the blue line) was also deemed successful. The red and black lines depict the mKate activation due to each protein in the absence of DOX. As expected, there was little to no fluorescence in these cases.

Exosome Isolation and Co-Culturing

Once we confirmed that the Acyl-TyA-rtTA fusion construct was functional, we progressed to exosomal experiments. We transfected Jurkat T cells with our constitutive Acyl-TyA-rtTA, cultured them, and isolated exosomes from them. Concurrently, we cultured HEK-293 cells transfected with our receiver circuits (rtTA-induced mKate and constitutive eBFP). Then, we added DOX and the isolated exosomes to the HEK cells. Initially, we observed a negative result—no mKate was detected—but we still have further optimization to do.

Cell-Cell Co-Culturing

Once we obtain a positive result from the exosome isolation experiment, we will move on to co-culturing the engineered Jurkat sender cells and HEK-293 receiver cells in a single dish. As shown in the graphic below, we expect the Jurkat cells to produce our fusion protein and package it into exosomes. The exosomes will then diffuse throughout the culture medium and, when they encounter the HEK cells, enter them and activate the mKate if DOX is also present.