We aim to construct a UV-responsive genetic switch that can initiate the expression of the target gene in the presence of UV-B signals and turn off the expression of the target gene when the UV-B signals are removed, while also controlling the expression level of the target gene to maintain it within an appropriate range. To achieve this, we have designed several components and four system pathways. The entire engineering process follows the DBTL (Design, Build, Test, Learn) principles recommended by iGEM, allowing for continuous transformation and iteration to enhance our project. Below are the four engineering cycles we have completed.

In order to verify the proper expression and potential interactions of UVR8, COP1, and RUP2 in human cells, we have designed an experiment to visualize these interactions using different colored fluorescent markers. We fused UVR8 with GFP, COP1 (which has a nuclear localization sequence) with mCherry, and placed RUP2 under the control of a tetracycline-inducible TRE promoter.

When the three plasmids are co-transfected, in the absence of UV-B signals, UVR8 forms a homodimer in the cytoplasm, and COP1 is localized in the nucleus, resulting in green fluorescence in the cytoplasm and red fluorescence in the nucleus. Upon exposure to UV-B signals, UVR8 monomerizes, enters the nucleus, and interacts with COP1, leading to the co-localization of red and green fluorescence in the nucleus. If tetracycline is added to induce RUP2 expression at this point, the dissociative effect of RUP2 may cause the co-localization in the nucleus to gradually disappear, with the green fluorescence fading. We aim to observe the changes in fluorescence within the nucleus before and after UV irradiation, as well as before and after tetracycline induction, thereby validating the interaction between UVR8 and COP1 in mammalian cells and the dissociation of their interaction by RUP2.

We ordered the plasmids pSNV2_CHER_NLS_COP1 and pIRESN2_GFP_UVR8 from Addgene, while pRP_TRE_RUP2 was synthesized by VectorBuilder.

We constructed the pRP_TRE_RUP2_Flag plasmid ourselves by adding a Flag tag to the end of the RUP2 gene using PCR. The plasmid was recombined and circularized through in vitro homologous recombination. Finally, we transformed it into DH5α to extract the pRP_TRE_RUP2_Flag plasmid.

We first performed colony PCR on the pRP_TRE_RUP2_Flag plasmid, and agarose gel electrophoresis showed the correct band. The gel image is as follows:

Additionally, we sent the constructed plasmid for sequencing, and the results confirmed that the sequence was entirely correct.

Subsequently, we conducted cellular experiments to verify the interactions of UVR8, COP1, and RUP2 in HEK293T cells.

To determine the optimal concentration of tetracycline for inducing RUP2 expression, we established a series of concentration gradients for testing. The pRP_TRE_RUP2_Flag plasmid was transfected into the 293T cell line, and Western blot analysis was performed to confirm the protein expression.

Plasmids pIRESN2_GFP_UVR8, pSNV2_CHER_NLS_COP1, and pRP_TRE_RUP2_Flag were simultaneously transfected into 293T cells. Cells were plated on cell climbing slices, and the three plasmids, numbered 1, 2, and 3, were co-transfected into the cells. After cultivation under conditions with and without tetracycline, observations were made using a confocal microscope, and images were captured. A field of view was located using 20x magnification. Subsequently, a field with more representative cells in different channels was identified, and a multi-channel film was prepared for cell counting.

We established a criterion for cell fluorescence. We counted the cells with cytoplasmic green fluorescence and no or weak nuclear green fluorescence. After observation, we counted these cells in multiple fields for group 1, group 2, and group 3 and performed statistical analysis. The results were as follows:

In addition, we implemented a time-lapse imaging approach to elucidate the subcellular dynamics and distribution of GFP-tagged UVR8 in response to UV-B irradiation. Our results revealed a significant enhancement in the nuclear GFP signal following UV-B exposure, suggesting the translocation of UVR8 into the nucleus upon UV-B stimulation.

In summary, we have verified that in mammalian cells, UV light can induce an interaction between UVR8 and COP1, while RUP2 can disrupt the interaction between UVR8 and COP1, successfully validating the interplay of UVR8, COP1, and RUP2.

In this cycle, we confirmed that UVR8 can translocate into the nucleus upon UV irradiation, and RUP2 plays a regulatory role in the nuclear import of UVR8. However, through Western blotting experiments, we quantitatively determined that the highest expression level of RUP2 protein was achieved with a tetracycline treatment concentration of 5 μg/mL. In contrast, in subsequent plasmid transfection fluorescence quantification experiments, we found that the proportion of cells with dark nuclear fluorescence after UV-B exposure was highest at a tetracycline treatment concentration of 10 μg/mL. This discrepancy may require further experiments to explore the optimal concentration of tetracycline for inducing RUP2 expression.

After validating the interactions of UVR8, COP1, and RUP2 in mammalian cells, we introduced the specific binding of GAL4 with 5xUAS and VP64 as a potent transcriptional activator to induce gene expression, constructing a UV-responsive gene switch and attempting to verify that the switch can be activated. We fused UVR8 with VP64, fused COP1 with a nuclear localization sequence and GAL4, and placed the GFP gene downstream of the 5xUAS sequence and the CMV promoter. Since COP1 is constantly localized in the nucleus, GAL4 within the nucleus will tightly bind to 5xUAS. Our GAL4 sequence utilizes the BD (DNA-binding domain), so the binding of GAL4 to 5xUAS will only serve a localization function and will not directly initiate gene expression. When UV-B signals are present, UVR8 monomerizes, enters the nucleus, and interacts with COP1, bringing VP64 closer to the CMV promoter and efficiently initiating the expression of the downstream GFP gene. We expect that upon exposure to UV-B, the cells will express green fluorescent protein, proving that our designed UV-responsive gene switch can be activated.

The GAL4 and VP64 genes were synthesized by Qingke, and the plasmid pDL6_5UAS_hCMVmin_SEAP_PA was provided by Professor Ye. We constructed the plasmids pIRESN2_Gal4_UVR8, pSNV2_VP64_NLS_COP1, and pDL6_5*UAS_hCMVmin_GFP ourselves.

We obtained the linearized vector and the target gene through PCR and used homologous recombination to assemble the target gene into the corresponding vector. The final constructs were verified by colony PCR and plasmid sequencing.

We first performed colony PCR on the plasmids pIRESN2_VP64_UVR8, pSNV2_GAL4_NLS_COP1, and pDL6_5*UAS_hCMVmin_GFP, and agarose gel electrophoresis showed the correct bands, with the gel images as follows:

Additionally, we sent the constructed plasmid for sequencing, and the results confirmed that the sequence was entirely correct.

Subsequently, we performed transfections in HEK293T cells, irradiated them with UV-B 24 hours post-transfection, and obtained results using an inverted fluorescence microscope 24 hours after UV-B irradiation.

In our experimental setup, a co-transfection approach was employed with the reporter gene plasmid in conjunction with the UVR8-VP64 plasmid and the COP1-NLS-Gal4 plasmid to form the experimental group. We established three control groups: one with solely the reporter gene plasmid, another with the reporter gene plasmid combined with the UVR8-VP64 plasmid, and a third with the reporter gene plasmid combined with the COP1-NLS-Gal4 plasmid. Furthermore, we included control groups to compare the effects of UV-B irradiation with those of non-irradiated conditions, thereby highlighting the regulatory role of UV-B within the system. A schematic representation of the transfection experimental design for both the experimental and control groups is depicted in the following figure 2：

We observed GFP fluorescence under 488nm excitation and 560nm emission using an inverted fluorescence microscope. We found that only the experimental group co-transfected with all three plasmids and irradiated with UV-B showed significant fluorescence, while all other groups showed no significant fluorescence.

For statistical data, we counted the cells with GFP fluorescence. After observation, we counted the cells in multiple areas of cells transfected with different plasmid combinations, calculated the number of fluorescent cells per unit area, and conducted a statistical analysis. The results are as follows:

We found that when three plasmids are co-transfected, exposure to ultraviolet light can significantly increase the fluorescence intensity within the cells, while there is almost no fluorescence intensity without UV exposure. At the same time, when transfecting two plasmids or a single plasmid, the fluorescence within the cells is barely observable regardless of whether UV light is exposed. This indicates that the gene switch we constructed strictly responds to ultraviolet light and has a high sensitivity.

In this cycle, we constructed and demonstrated the feasibility of the UV-B-activated reporter gene transcription system using a GFP reporter gene. Our study establishes a correlation between UV-B irradiation and the initiation of gene expression by demonstrating the pivotal role of "UVR8 nuclear entry." Furthermore, the successful expression of GFP under specific conditions confirms the system's capability to discern between environments with and without UV-B exposure. This robust evidence underscores the viability of employing UV-B as a trigger for gene expression.

After verifying that the UV-responsive gene switch we constructed can be activated, we connected the RUP2 gene downstream of the target gene using the P2A sequence. P2A is a self-cleaving peptide that allows the genes before and after the sequence to be translated independently. The addition of RUP2 can, to some extent, inhibit the overexpression of the target gene when the UV-B signal activates the gene switch.

To quantitatively characterize RUP2's regulation of the target gene's expression, we used the luciferase gene as the target gene and connected the RUP2 gene downstream of the luciferase gene via the P2A sequence. We attempted to construct two types of plasmids with the luciferase gene as the target gene, one with RUP2 and one without RUP2. After transfecting cells with the two plasmids and exposing them to UV-B radiation for a certain period, we measured the luminescence intensity of luciferase and luciferin. This allowed us to assess the expression level of the target luciferase gene and quantitatively characterize the effect of RUP2 on the expression level of the target gene.

The luciferase gene was provided by Professor Ye.

We constructed the plasmids pDL6_5UAS_hCMVmin_Luciferase and pDL6_5UAS_hCMVmin_Luciferase_P2A_RUP2 ourselves. We obtained the linearized vector and the target gene through PCR and used homologous recombination to assemble the target gene into the corresponding vector. After obtaining the pDL6_5xUAS_hCMVmin_Luciferase_P2A_RUP2 plasmid, we further deleted the P2A_RUP2 sequence through PCR and obtained the pDL6_5xUAS_hCMVmin_Luciferase plasmid by in vitro recombination. The final constructs were verified by colony PCR and plasmid sequencing.

We first performed colony PCR on the plasmids pIRESN2_VP64_UVR8, pSNV2_GAL4_NLS_COP1, and pDL6_5*UAS_hCMVmin_GFP, and agarose gel electrophoresis showed the correct bands, with the gel images as follows:

Additionally, we sent the constructed plasmid for sequencing, and the results confirmed that the sequence was entirely correct.

Subsequently, we performed transfections in HEK293T cells, irradiated them with UV-B 24 hours post-transfection, and obtained results using an inverted fluorescence microscope 24 hours after UV-B irradiation.

After a certain period of protein expression, we lysed the cells and added a luciferin substrate to measure the activity of luciferase.

As illustrated in the figure 27, the impact of UV-B irradiation on the expression of the luciferase reporter gene in the complete system containing three genetic elements is significant. After exposure to UV-B, the expression of the reporter gene increased to nearly five times that of the original, indicating that the gene system we designed is capable of sensitively responding to UV-B input and using the expression of the reporter gene as an output. In other groups containing only two genetic elements or just the reporter gene plasmid, the presence or absence of UV-B does not significantly affect the expression of the reporter gene. This demonstrates that each element in the system is indispensable, and also solidifies the experimental results through the control.

The results above show that the gene switch we constructed does indeed respond strictly to ultraviolet light and with high intensity. Subsequently, using the same experimental procedures, we obtained the luminescence intensity statistics of the plasmid of pDL6_5xUAS_hCMVmin_Luciferase_P2A_RUP2.

By analyzing the results of the plasmid pDL6_5xUAS_hCMVmin_Luciferase_P2A_RUP2, we found that there is no significant difference in luminescence intensity between co-transfection of all three plasmids and transfection of any two plasmids.

In this cycle, we found that in the system without RUP2, co-transfection of all three plasmids greatly increased the expression of luciferase under UV induction. However, for the system containing RUP2, there was no significant difference in the luminescence intensity between co-transfection of all three plasmids and transfection of any two plasmids after UV induction. We speculate that this might be due to the high expression level of RUP2, which strongly inhibits the interaction between UVR8 and COP1, resulting in almost no efficient initiation of luciferase gene expression. Therefore, in the future, we will reduce the expression level and rate of RUP2 by increasing the number of rare codons on RUP2, so that the final expression level of the target gene stabilizes at an appropriate concentration.