EL222 is a natural photosensitive DNA-binding protein that dimerizes and binds DNA upon blue light exposure. It is composed of a N-terminal light-oxygen-voltage (LOV) domain and a helix-turn-helix DNA-binding domain at the C-terminus. EL222 could act as a transcriptional activator in a tunable blue light-inducible promoter system. pBlind, a synthetic blue light-inducible promoter, is formed by replacing the lux box (LuxR and 3-oxo-C6-HSL complex binding region) which is a 20-bp inverted repeat from the luxI promoter with the 18-bp EL222 binding protein region. When exposed to blue light, EL222 dimerizes and overlaps the -35 region of the promoter thus recruiting RNAP (RNA polymerase). Because the LOV domain is in equilibrium between the dimer and the monomer in the dark state, a high leakage level of this blue light-induced system is witnessed. Aiming to improve the issue, XMU-China 2021 has replaced the strong promoter BBa_J23106 with pBlind. While progress was made, some reporter genes still expressed without blue light.

LicV is a fusion protein, which could be activated with blue light. It consists of the N-terminal RNA-binding domain (coantiterminator, CAT) derived from LicT of Bacillus subtilis and Vivid (VVD) which is a small LOV domain-containing protein. When induced by blue light, LicV undergoes structural alterations that enhance the stability of its dimeric form and selectively attaches to the ribonucleic antiterminator (RAT) RNA sequence, effectively inhibiting the formation of an RNA terminator stem-loop structure.

This year, NAU-CHINA tried to resolve the leakage of the system and enable its function as intended.

Fig. 1 | EL222 system designed by XMU-China 2021.

This year, the improvement of NAU-CHINA is to lower the leakage level of the BBa_K3739064 at the level of transcription. Aiming to resolve the leakage issue, we embedded RAT-Terminator between the pBlind promoter and superfolder (sfGFP) reporter gene. Besides, the expression level of LicV is high enough to ensure that the sfGFP can be transcribed normally due to the strong promoter J23104. When in the absence of blue light, an RNA terminator stem-loop structure is formed that the transcription is terminated. And while activated by blue light, LicV dimer forms and binds to a ribonucleic antiterminator (RAT) RNA sequence to prevent the termination of transcription.

Fig. 2 | Improved EL222 system with LicV and RAT.

The pBlind-EL222-B0015-pBlind-sfGFP-rrnB fragments were inserted into the pCDFDuet-1 vector whose promoter and multiple cloning site (MCS) were removed. And the J23104-LicT^CAT-linker-VVD-rrnB fragments were inserted into the pQE-60 vector to express LicV.

To characterize the improved EL222 blue light-inducible system and evaluate its reduction in leakage, we designed four experimental groups, each containing or lacking the RAT terminator and LicV fusion protein genes. Then we co-transformed these plasmids into E. coli BL21(DE3) and cultured bacteria solution at 37 °C, 200 rpm for 12 h under blue light (460nm) or darkness, took 100 μL of culture every 2 h for detection of fluorescent intensity and OD₆₀₀.

The experimental setup includes an experimental group (RAT+ LicV+) and three control groups: one containing RAT but lacking LicV (RAT+ LicV-), one containing LicV but without RAT (RAT- LicV+), and a blank group without either component (RAT- LicV-). The detailed structures, expected outcomes, and experimental conditions of each group are illustrated in the accompanying figures (Fig. 3).

Fig. 3 | Experimental Setup and Group Descriptions of each group.

a.Experimental Group (RAT+ LicV+): Contains both RAT and LicV, showing minimal leakage under dark conditions and sfGFP induction under blue light. b.Control Group 1 (RAT+ LicV-): Contains RAT only, with minimal sfGFP expression regardless of illumination. c. Control Group 2 (RAT- LicV+): Contains LicV only, showing increased leakage without light due to the absence of RAT regulation. d.Control Group 3 (RAT- LicV-): Lacks both regulatory elements, with the highest leakage observed.

All four groups were subjected to both blue light exposure and dark conditions during cultivation, leading to a total of eight experimental setups. Bacteria were cultured at 37°C, and samples were taken every 2 h over a 16-hour period for fluorescence and OD₆₀₀ measurements by PerkinElmer Ensight multimode plate reader.

Table 1 | Expression conditions and expected results of each group.

The fluorescence intensity of sfGFP was measured at multiple time points across the 16-hour period under both blue light and dark conditions, and the results revealed distinct patterns in gene expression across the different groups. Under dark conditions, the experimental group (RAT+ LicV+) exhibited significantly lower fluorescence compared to Control Group 3 (RAT- LicV-), indicating that the inclusion of the RAT element effectively reduced system leakage in the absence of light. Specifically, the experimental group showed a 23.05% reduction in fluorescence leakage compared to the blank control group, highlighting the success of the RAT element and LicV protein in suppressing background expression when blue light is not present.

Control Group 1 (RAT+ LicV-) displayed consistently low fluorescence under both light and dark conditions, demonstrating that the RAT element alone was sufficient to prevent gene expression, regardless of illumination. This suggests that without the presence of LicV, the downstream gene expression remained effectively inhibited. In contrast, Control Group 2 (RAT- LicV+) showed fluorescence levels similar to those of Control Group 3 under both light and dark conditions, indicating that, in the absence of the RAT sequence, LicV was unable to perform its regulatory function. As a result, gene expression remained uncontrolled, leading to similar fluorescence levels as observed in the fully unregulated system of Control Group 3.

Furthermore, both the experimental group and Control Group 2 exhibited lower fluorescence than Control Group 3 under blue light, suggesting that the introduction of additional regulatory elements, such as the RAT terminator and the LicV protein, may have imposed a metabolic burden that impacted the efficiency of gene expression. Despite this burden, the significant reduction in fluorescence under dark conditions for the experimental group demonstrates that our modifications successfully reduced the background expression, thereby enhancing the overall controllability of the system.

Fig. 4 | Fluorescence intensity over time for all groups.

Comparison of fluorescence intensity changes over time among different groups.BL21(DE3) served as an untransformed E. coli blank control. Comparison between a. the experimental group and control group 1. b. the experimental group and control group 2. c. the experimental group and control group 3. d. control group 2 and control group 3.

The microscopy images align well with the trends observed in the enzyme-linked measurements, demonstrating the regulatory efficiency of our constructed system. Figure 1 shows that under blue light, our system effectively induced the expression of the fluorescent protein, resulting in strong fluorescence. Figure 2 demonstrates that in the experimental group under dark conditions, the expression of the fluorescent protein was well controlled, showing minimal fluorescence leakage. Figure 3 indicates that in the absence of LicV, the RAT element effectively suppressed downstream gene expression, as evidenced by the very low fluorescence signal. In Figure 4, with both LicV and RAT absent, the fluorescence protein expression was significantly higher, likely due to reduced metabolic burden, leading to more extensive fluorescence. These results are consistent with the data obtained from the microplate reader, further validating the role of RAT and LicV in regulating gene expression and minimizing leakage.

Fig. 5 | Fluorescence microscopy images of different groups.

a.Fluorescence expression of the experimental group under blue light exposure. b.Fluorescence expression of the experimental group in darkness. c. Fluorescence expression of control group 1 under blue light exposure. d.Fluorescence expression of control group 3 under blue light exposure.