Developing Novel Kinase Activity Reporters Utilizing Bioluminescence

Overview

Biosensors are an innovative technology that use fluorescence to visualize protein expression and activity in eukaryotic cells primarily using a Forster Resonance Energy Transfer (FRET) system. This project focuses on the hypothesis that a Bioluminescence Resonance Energy Transfer (BRET) pair, using Nanoluc as the donor, can be used as a kinase activity reporting unit when combined with molecular switches that are dependent on said kinase activity. I created a BRET assay by bonding a donor luciferase protein and acceptor protein to form a substrate couple that was transfected into HEK293T cells and then imaged under a microscope and plate-reader in order to quantify the bioluminescence. ​  By taking Nanoluc as the donor luciferase and utilizing various substrates coupled to PKA, PKC, Src, and ERK I was able to develop 4 viable biosensors. The PKA biosensor had a signal increase of 100%, PKC had a signal increase of 20%, ERK had a signal increase of 50%, and most notably Src had a signal increase of 90%, 9x more than any previous Src activity reporter made using BRET.​  BRET-based biosensors make biosensor technology much more inexpensive, due to the plate-reader based assay rather than the expensive microscope that need to be utilized with FRET biosensors, making these results significant. I was able to develop working biosensors with strong signals during the first round of bioengineering. Some next steps involve optimizing the sensors by changing variables such as linker length, as well as targeting the biosensors to specific sub-cellular organelles.​

Competition history

• CSEF 2026 Biochemistry/ Molecular Biology (Senior Division) · Entry S-04-15

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