From 2013.igem.org

Overview

Our rtTA fusion proteins are crucial for one method of gene activation in our sender cells. Here, we employed the highly oligomeric cytoplasmic protein TyA with an N-terminal acylation tag (MGCINSKRKD-) and, later, a polyhistine tag to target proteins to exosomes. In this case, the transported protein is a reverse tetracycline-controlled transactivator (rtTA3, here referred to as rtTA) that must be sent to exosomes for export so that it can activate genes in receiver cells. In the presence of doxycycline (DOX), rtTA can be used to activate the tetracycline response element (Tre-tight, or Tre-t) promoter, driving the expression of previously inactive genes.

To test the functionality of rtTA in single cells, a constitutive expression of rtTA was driven by the human elongation factor 1α (hEF1α) promoter. A receiver circuit consisting of mKate fused to the Tre-t promoter was also constructed. In theory, the constitutive rtTA would drive the expression of mKate when we added DOX. Jurkat T cells were nucleofected with both plasmids and incubated with DOX. The mKate fluorescence was observed 48 hours post nucleofection, proving that the rtTA worked. Then, we assembled an rtTA fusion protein - Acyl-TyA-rtTA.

Overall, rtTA is a very useful part because it can be easily toggled on and off with DOX. Here we confirm that it can be fused to exosome-targeting sequences and still retain its functionality. Although we only activate mKate here, rtTA does not apply just to fluorescent proteins - it can possibly be used to activate any gene driven by the Tre-t promoter.

Characterization

Since it is such a crucial component in many of our exosomal cargoes, rtTA needed to be tested before we could attempt to send it. In our project, rtTA is used in conjunction with 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.

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-t 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 DOX.

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.

Western Blot

While sending proteins into exosomes is one of the major objectives of our project, the story is not complete without the detection of the proteins: the amount in the sender cells transfected with the desired fusion gene, the amount of protein exported to the membrane and into exosomes, and the amount of protein that ultimately traverses across a synapse within exosomes and reaches receiver cells. Sending fluorescent proteins allows us to check for their presence through confocal imaging but we had a variety of other proteins attached to our Acyl-TyA fusions that could not simply be imaged. We needed a more conclusive method of detecting and eventually relatively quantifying where our protein was going after being produced in the sender cells.

Since we have a wide variety of proteins, we wanted to add a common component to each fusion so we would not require a multitude of primary and secondary antibodies. Therefore, we added a His Tag (a small short amino acid repeat sequence) at the C terminus of the Acyl-TyA-mKate fusion.

Fifteen wells were prepared: the first five were a gradient of HEK cell lysate (0.5 ul to 12 ul of protein), the next 8 were a gradient of HEK cell lysate transfected with Acyl-TyA-mKate-His fusion (0.5-12 ul of protein). The last well was for the protein in the media of our Acyl-TyA-mKate-His fusion (12 ul).

The primary antibodies that were used were Anti-GADPH and Anti-His. In order to image the membrane using the Odyssey CLx we used IR dye Secondary antibodies (800 CW and 680 RD).

After completing the required protocol, the following was the image of the gel (Wells arranged from 1-15)

The green signal on the blot corresponds to the detection of GADPH at around 37 kDA. The red signals in the right most well correspond to the detection of the His tag. As observed, all of the wells contained GADPH meaning that all of the wells contained HEK Cell lysates. The brightness of the signal corresponds to the amount of protein present in each well and varies due to the varied amounts of lysate in the wells.

After affirming our positive control, we see that the His Tag is only present in the well containing our Fusion-transfected HEK cell lysate media. There is no His Tag in the Fusion-Transfected HEK cell lysate wells. Based on these results, we hypothesize that our Acyl-TyA-mKate-His fusion was exported out of the cell and into the media.

Based on these positive results, we are going to be conducting future Western Blots to establish that the proteins being exported into the media are leaving contained within exosomes. We have an Anti-CD63 antibody that targets CD63 protein, a protein ubiquitously present on the membrane of exosomes . Using the Anti-CD63 and Anti-His, we are going to isolate exosomes and run a western blot with the exosome lysate that hopefully contains our mKate protein to check for the detection of mKate with our isolated exosomes.

Once we confirm that the mKate is in the media and within exosomes, our future goals are going to be to add His Tags to more of our fusions or to use different primary and secondary antibodies to test for the presence of rtTA3, L7Ae etc.

Exosome Isolation and Application

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.