Design
In the design phase, we wanted to create a mammalian cell-based biosensor that could detect oxidative and electrophilic stress caused by harmful cosmetic ingredients such as hydroquinone. To design our biosensor, we drew inspiration from the Keap1-Nrf2 signaling mechanism, a key cellular defense pathway against oxidative and electrophilic stress. Under normal conditions, Nrf2 is bound by Keap1 and targeted for degradation; however, during oxidative stress, Keap1's cysteine residues are modified by electrophiles and ROS, releasing Nrf2 to translocate into the nucleus. There, it binds to specific DNA sequences known as Antioxidant Response Elements (AREs) located upstream of stress-responsive genes (Figure 1).
We harnessed this mechanism by placing an ARE enhancer sequence upstream of a minimal CMV promoter to create an inducible transcriptional system (Figure 2). When Nrf2 binds to the ARE, it initiates transcription of our EGFP reporter gene, producing a measurable fluorescence signal in response to oxidative stress (Figure 3).
Figure 1. Keap1-Nrf2 signaling mechanism under normal and oxidative stress conditions
Figure 2. The initial plan of our plasmid construct
Figure 3. The proof-of-concept of our biosensor
Build
We built our construct in silico using SnapGene. We modified the existing CMV enhancer-promoter part by removing the enhancer region and inserting the ARE sequence to create the ARE-minCMV-EGFP construct. The designed fragment was then virtually assembled into the pGGAselect mammalian vector.
Figure 4. In silico assembly of ARE-minCMV-EGFP construct in SnapGene
Figure 5. Final construct in pGGAselect mammalian vector
TEST & LEARN
Although we were unable to perform wet-lab testing due to time and resource limitations, we validated our design through in silico and literature review. Prior studies have shown that Nrf2 activation by oxidative agents increases transcription of ARE-driven genes, supporting the expected functionality of our construct.
Through this process, we learned about the complexity of mammalian gene regulation, the importance of modular design, and biosafety considerations in synthetic biology. These insights will guide the next stage, where we plan to experimentally express and analyze the ARE-minCMV-EGFP plasmid in HEK293T cells to confirm its reporter activity.