The YF1, FixJ and Fixk2 promoter can constitute a blue light-regulated system. In the absence of blue light, YF1 phosphorylates its cognate response regulator FixJ, which then drives robust gene expression from the Fixk2 promoter. Upon light absorption, net kinase activity of YF1 and consequently gene expression are greatly reduced^[1].

However, practical implementation of this optogenetic circuit has faced challenges. For instance, CAU-China 2022 (BBa_K4192130) was unable to achieve normal expression and detect mCherry reporter signals due to its weak promoter. Additionally, the blue light-regulated system composed of YF1, FixJ and Fixk2 promoter exhibited significant leakage issues, which results in a relatively small dynamic range of blue light response for this optogenetic circuit. Although NAU-CHINA 2023 (BBa_K4613462) improved this by using a T7 promoter and a gfp reporter gene, the key PAL sequence was not verified due to time constraints.

To address the issue of the dynamic range of the light response in the blue light-regulated system, one promising strategy involves incorporating a second layer of regulatory control. The lactose operon induction system, commonly used in Escherichia coli, relies on the binding of lactose or isopropyl β-D-1-thiogalactopyranoside (IPTG) to the lactose repressor protein LacI, playing a pivotal role in controlling the lactose operon. In the absence of the chemical inducer IPTG, the repressor protein LacI binds to the operator sequence lacO, effectively blocking the RNA polymerase from transcribing the target genes. By modifying the lactose repressor protein LacI through the light-oxygen-voltage 2 (LOV2) domain of the photosensitive element, the OptoLacI^D protein was obtained, which responds to blue light instead of IPTG^[2]. This means that under the combined action of the OptoLacI^D protein and LacO, it is possible to improve the dynamic range problem of the blue light-regulated system by mediating the transcriptional expression of the reporter gene gfp.

This year, NAU-CHINA 2025 tried to provide a new solution for increasing the dynamic range of the light response in the blue light-regulated system and enable its function as intended.

Figure 1. Light control system designed by CAU-China 2022.

Figure 2. Light control system designed by NAU-CHINA 2023.

Aiming to enhance the system's ability to distinguish between blue light and dark signals, we integrated OptoLacI^D (BBa_25BHXP3N) into the plasmid and embedded the operator sequence lacO3 (BBa_25V87BRR) between the Fixk2 promoter and gfp reporter gene. Under blue light irradiation, OptoLacI^D can bind to the operator sequence lacO3, effectively blocking the RNA polymerase from transcribing the target genes and achieving the goal of expanding the dynamic range of the light response.

Figure 3. Improved blue light-regulated system with OptoLacI^D and LacO3.

To characterize the improved blue light-regulated system and evaluate the dynamic range of the blue light response, we designed an experimental group and a control group to compare with NAU-CHINA 2023. Then we transformed these plasmids into Escherichia coli BL21(DE3) and precultured bacteria solution at 37°C, 200 rpm for 8 h under blue light (460 nm) or darkness. Subsequently, the cultures were inoculated into 1 mL LB medium supplemented with kanamycin, at a 1% inoculum, in two 24-well plates. One plate was incubated at 37°C under dark condition, while another plate was incubated at 37°C with blue light irradiation, taking 100 μL of culture into 96-well plates every 2 h for detection of fluorescent intensity and OD₆₀₀. For GFP fluorescence signal measurement, the SpectraMax iD5 microplate absorbance reader with an excitation wavelength of 488 nm and an emission wavelength of 530 nm was utilized.

The experimental setup includes an experimental group (OptoLacI^D+ LacO3+) and a control groups (OptoLacI^D- LacO3-). Both groups were subjected to both blue light exposure and dark conditions during cultivation, leading to a total of four experimental setups. Bacteria were cultured at 37°C, and samples were taken every 2 h over a 12-hour period for fluorescence and OD₆₀₀ measurements.

Figure 4. The gene pathways of the experimental group and the control group.

The fluorescence intensity of GFP was measured at multiple time points across the 12-hour period under both blue light and dark conditions, and the results revealed distinct patterns in gene expression across the different groups. The control group exhibited the expected light response trend: the fluorescence intensity under dark conditions was higher than that under blue light conditions. However, the difference between the two in the control group was small, indicating that the dynamic range of the light response is limited.

After introducing OptoLacI^D and LacO3, the most prominent effect of the experimental group was a significant increase in the difference in fluorescence intensity under dark and blue light conditions. The expression of the gfp gene under dark conditions was strongly activated, with the average fluorescence intensity being approximately twice that of the control group. At the same time, although the fluorescence expression of the control group was still inhibited under blue light, the residual fluorescence intensity after inhibition was slightly higher than that of the control group.

Figure 5. Fluorescence intensity over time for all groups.

Within a short period of time (2-6 h), the difference in fluorescence intensity between the experimental group under dark and blue light conditions was significantly increased, compared with the control group. This result confirmed that OptoLacI^D and LacO3 significantly enhanced the system's ability to distinguish between blue light and dark signals. In conclusion, although OptoLacI^D and LacO3 changed the absolute expression level of the blue light-regulated system, they successfully achieved the core design goal of expanding the light response dynamic range by super-proportionally enhancing the dark-induced expression.

Figure 6. Fluorescence intensity differences between each group.
Multiple unpaired t tests; *P<0.05, **P<0.01, ***P<0.001.

Our improvement achieved the optimization of signal-to-noise ratio by significantly enhancing the maximum output expression of the system in the "on" state. This "enhanced signal" strategy has unique advantages in many applications. For situations requiring high-level protein production, a system with an extremely high induction expression level is the primary requirement. The peak expression level that our system can achieve under dark conditions is much higher than that of the original system, providing a foundation for efficient production. In sensing and imaging applications where signal-to-noise ratio is of critical importance, although the absolute background has increased, the greatly enhanced signal makes it stand out in the background and becomes easier to detect and quantify. This is similar to using intense excitation light in optical detection to overcome background fluorescence. Future work can focus on integrating the powerful expression enhancement module (OptoLacI^D and LacO3) that we have developed this time with the next-generation inhibition module that can more thoroughly "switch off" the system, ultimately achieving an ideal system that simultaneously possesses ultra-high expression and ultra-low background.

• [1]Ohlendorf R, Vidavski R R, Eldar A, et al. From dusk till dawn: one-plasmid systems for light-regulated gene expression[J]. Journal of Molecular Biology, 2012, 416(4): 534-542.
• [2]Liu M, Li Z, Huang J, et al. OptoLacI: optogenetically engineered lactose operon repressor LacI responsive to light instead of IPTG[J]. Nucleic Acids Research, 2024, 52(13): 8003-8016.