Results
Preview
After we decided on two vesicles and figured theoretically out how we want to load our vesicles specifically for our non-invasive cell monitoring approach we performed the experiments necessary to proof the functionality of our system. For those who are interested in the exact constructions of our vesicles can find a detailed plan at “Model”. In short we use the following two vesicle types:
1. Exosomes: a naturally occurring secretion pathway of cells to get rid of different materials like cellular trash but also valuable information for other cells.
2.Virus-like particles (VLPs): artificially produced vesicle type that can be used as extracellular vesicle to export information.
Our vesicles are non-toxic and can load very specific information depending on what we want to analyze and monitor.
Transmission electron microscopy
To check that our vesicles are there and intact, they were first purified via single-chain Avidin-Biotin interactions or His-Tag purification. After that, the VLPs and exosomes were prepared for transmission electron microscopy (TEM). TEM is a microscopy technique where a high energy beam of electrons is shown through a very thin sample fixed on a grid.
In the two figures below the exosomes (left) and VLPs (right) can be seen. Furthermore, with this technique, it is possible to calculate the diameter of our vesicles. For the exosomes, the diameter is xx nm which is in the range of average exosome diameter which ranges from, for the VLPs it is xx nm.
Split luciferase assay
To validate the formation and export efficiency of our synthesized exosomes and VLPs we fused an HiBiT-Peptidetag to our constructs (see basic parts). A HiBiT-Tag is one half of a luciferase. If we add the second part, the polypeptide LgBiT, both form spontaneously a functional luciferase called NanoBiT enzyme which glows after substrate addition[1].
The luminescence was measured via a plate reader. To quantify the amount of measured HiBiT luminescence in the cells and the supernatant (that contain Exosomes or VLPs) a standard curve has been made. Because the cell and the supernatant samples were measured on two different plates, two standard curves were necessary (see Appendix).
[1] https://www.promega.de/products/protein-quantitation-and-detection/protein-quantitation/nano-glo-hibit-lytic-detection-system/?catNum=N3030
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Video
| Materials | Price for a single reaction (15µl) | Price per patient |
|---|---|---|
| Buffer + Media | $0.19 | $0.13 |
| Energy Solution | $2.99 | $1.99 |
| Phage DNA | $0.07 | $0.05 |
| Work | $0.76 | $0.50 |
| Total | $4.01 | $2.67 |
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