Proof of Concept

Validation of the Feasibility and Reversibility of the Light-Responsive System

To validate that the two primary optogenetic components of the LEGO system, iLID and sspB, exhibit consistent and stable nuclear localization under blue light illumination, we fused each protein with a nuclear localization sequence (NLS). Both short-term (200 s) continuous blue light stimulation and long-term (18 h) pulsed blue light illumination resulted in robust nuclear localization of the components, demonstrating the stability and reliability of this light-responsive system (Figure 1).

To visually validate the blue light-responsive interaction between the optogenetic components, their spatial redistribution was monitored. The sspB protein was anchored to the plasma membrane via a membrane localization sequence (NLYN), while iLID remained freely diffusible in the cytoplasm. Upon brief blue light illumination (100 s), iLID was rapidly recruited to the membrane (Figure 2). After blue light withdrawal, iLID quickly returned to its diffuse cytoplasmic distribution in the dark, demonstrating the reversibility of this optogenetic module.

Validation of Bioluminescence-Induced Activation of the Light-Responsive System

We next sought to determine whether bioluminescence generated by NanoLuc (Nluc) could activate the iLID-sspB interaction. Upon adding the Nluc substrate fluorofurimazine (FFz) to the culture medium, we observed robust recruitment of iLID to the plasma membrane, closely mirroring the localization pattern induced by blue light illumination (Figure 3). These results validate the feasibility of using intracellular bioluminescence to activate the light-responsive components within the LEGO system.

Validation of Light-Activated Co-expression of Insulin and GLP-1

Using live-cell imaging and quantitative real-time PCR, we confirmed the feasibility and light responsiveness of the LEGO system in activating target gene expression. After 24 hours of pulsed blue light illumination, a significant increase in co-expressed mCherry fluorescence was observed. Consistently, the mRNA levels of Insulin (INS), GLP-1, and mCherry were markedly upregulated compared to the dark control group (Figure 4). These results demonstrate that the LEGO system enables robust, blue light-dependent co-expression of Insulin and GLP-1.

Validation of Engineered Human Insulin Activity

To confirm the secretion and bioactivity of insulin produced by our engineered LEGO system, we employed live-cell imaging to monitor the activation of the PI3K-Akt signaling pathway in response to conditioned medium from HEK-293T cells expressing the LEGO system. Culture medium collected from cells exposed to either blue light stimulation or dark incubation was applied to reporter cells expressing AktPH-EGFP (the PH domain of Akt fused with EGFP). We observed significant translocation of AktPH to the plasma membrane in cells treated with medium from blue light-stimulated conditions, confirming blue light-dependent production, secretion, and bioactivity of the insulin generated by the engineered LEGO gene circuits (Figure 5).