In 2024, we handed in 25 documented bio-brick parts, including all those we used in our project.

Negative Feedback Regulatory Component - BBa_K5182003:RUP2 (repressor of UV-B photomorphogenesis 2)

Our favorite Basic part is BBa_K5182003 (RUP2). This part mainly serves a negative feedback role in the system. RUP2 is a core protein in plants that regulates the response to ultraviolet B (UV-B). As a negative regulatory factor in the UV-B signaling pathway, RUP2 can competitively bind to UVR8 with COP1, thereby dissociating UVR8 from the UVR8-COP1-SPA4 complex, promoting the re-dimerization of UVR8 and resulting in the loss of its activity. This mechanism limits the intensity and duration of the response. Such precise regulatory mechanisms can be applied to human cells to control the expression of the XPC gene in gene therapy.

Optogenetic system validation module:

Our favorite composite parts mainly include BBa_K5182021 (UVR8-GFP), COP1-NLS-mcherry (BBa_K5182022), and TRE-RUP2 (BBa_K5182023). By using these three composite parts, we can monitor the dynamic interactions between UVR8 and COP1 through the observation of changes in fluorescence localization and intensity. Additionally, we can induce the production of RUP2 using tetracycline to test whether our optogenetic system has a negative feedback regulatory mechanism.

1. Wang Y, Wang L, Guan Z, et al. Structural insight into UV-B–activated UVR8 bound to COP1. Science Advances, 2022, 8(16): eabn3337.
2. Rizzini, L., Favory, J. J., Cloix, C., Faggionato, D., O’hara, A., Kaiserli, E., ... & Ulm, R. (2011). Perception of UV-B by the Arabidopsis UVR8 protein. Science, 332(6025), 103-106.
3. Jenkins, G. I. (2014). The UV-B photoreceptor UVR8: from structure to physiology. The Plant Cell, 26(1), 21-37.
4. Shimizu-Sato, S., Huq, E., Tepperman, J. M., & Quail, P. H. (2002). A light-switchable gene promoter system. Nature Biotechnology, 20(10), 1041-1044.
5. Yin, R., & Ulm, R. (2017). How plants cope with UV-B: from perception to response. Current Opinion in Plant Biology, 37, 42-48.

RUP2 is a core protein in plants that regulates the response to UV-B radiation, serving as a negative regulator in the UV-B signaling pathway. It can dissociate the interaction between UVR8 and COP1, promote the re-dimerization and inactivation of UVR8, and terminate a series of downstream signal responses triggered by the combination of UVR8 and COP1 induced by UV irradiation.

In order to be able to improve the work efficiency and biosafety of the photogenetic system, we employed two special designs. First, we used a truncated version of RUP2, as previous studies have demonstrated that the core functional protein of RUP2 is WD40. Directly using the gene sequence of the RUP2 core protein WD40 will help improve the expression efficiency of this BioBrick.

At the same time, to verify the negative feedback regulation of RUP2 in optogenetic systems，we introduced the RUP2 encoding gene into the plasmid of pRP_TRE_RUP2_Flag and obtained a tetracycline induced expression of RUP2 protein plasmid.

We first validated the negative feedback regulation of RUP2 on the UVB response system UVR8-COP1 in mammalian cells. This enabled us to establish a UVB signal-regulated, reusable optogenetic system, providing a targeted control tool for gene therapy. By controlling the ultraviolet irradiation area, we can limit gene expression to damaged cells or specific skin areas that require treatment. This localized expression control can enhance therapeutic efficacy while avoiding unnecessary effects on other cells in the body.

More importantly, our basic part RUP2 provides a natural negative feedback regulation mechanism for the system, preventing excessive activation of exogenous gene expression. This ensures that gene expression can be timely shut down after the light signal disappears, preventing adverse reactions in cells due to overexpression of genes.

1. Wang Y, Wang L, Guan Z, et al. Structural insight into UV-B–activated UVR8 bound to COP1. Science Advances, 2022, 8(16): eabn3337.
2. Rizzini, L., Favory, J. J., Cloix, C., Faggionato, D., O’hara, A., Kaiserli, E., ... & Ulm, R. (2011). Perception of UV-B by the Arabidopsis UVR8 protein. Science, 332(6025), 103-106.
3. Jenkins, G. I. (2014). The UV-B photoreceptor UVR8: from structure to physiology. The Plant Cell, 26(1), 21-37.
4. Shimizu-Sato, S., Huq, E., Tepperman, J. M., & Quail, P. H. (2002). A light-switchable gene promoter system. Nature Biotechnology, 20(10), 1041-1044.
5. Yin, R., & Ulm, R. (2017). How plants cope with UV-B: from perception to response. Current Opinion in Plant Biology, 37, 42-48.

To verify the feasibility of the UVB light-sensing system UVR8-COP1-RUP2 in mammalian cells, we designed a set of fluorescence labeling experiments to monitor the dynamic behavior of UVR8 and COP1 under UV-B treatment in real time through fluorescence signal changes. In this project, the UVR8 protein is fused with green fluorescent protein (GFP), and the COP1 protein is fused with a nuclear localization signal (NLS) and red fluorescent protein (mCherry). The RUP2 gene from plants is connected to a tetracycline-inducible promoter (TRE promoter). Together, they form our three favorite composite parts: UVR8-GFP (BBa_K5182021), COP1-NLS-mcherry (BBa_K5182022), and TRE-RUP2 (BBa_K5182023).

Using these three composite parts, we successfully validated the interaction between UVR8 and COP1 in mammalian cells after receiving UVB signals. Additionally, upon releasing tetracycline to induce RUP2 production in the environment, we observed the negative feedback regulation of RUP2 in the optogenetic system.

We have created a novel UVB signal-responsive system with negative feedback regulation, providing new insights for the controllability and effectiveness of gene therapy for genetic diseases, particularly suitable for complex diseases requiring precise gene expression control. Similarly, our optogenetic system is also applicable to scenarios such as the construction of light-controlled cell factories in synthetic biology.

To verify the negative feedback regulation of RUP2 in optogenetic systems，we introduced the RUP2 encoding gene into the plasmid of pRP_TRE_RUP2_Flag and obtained a tetracycline induced expression of RUP2 protein plasmid.Based on the experimental design, RUP2 can competitively bind to UVR8, thereby preventing the interaction between UVR8 and COP1. Once RUP2 binds to UVR8, the yellow light signal from the co-localization of UVR8 and COP1 in the nucleus will gradually fade. This process allows the role of RUP2 in competing for the binding of UVR8 and COP1 to be visually reflected by changes in yellow light intensity.

In our previous pre-experiment, By Western blot we verified that RUP2 can be inducibly expressed under the control of Tet-on promoter, and we obtained the optimal concentration of tetracycline induction to be 5 μg/ml by setting the concentration gradient, that is, the working concentration is 5 μg/ml.

In order to verify the mechanism of action of the three composite parts of the whole optogenetic system, we subjected the plasmids pIRESN2-GFP-UVR8, pSNV2-CHER-NLS-COP1 and Tet-on-RUP2 to different treatments on 293t cells (Table 1)

We performed the transfection experiments according to the following time line: we irradiated the well plates with UV light at 100 J/m2 at 24 hours after transfection, and then we added tetracycline to each well at a concentration of 5-10 μg/ml at 29 hours after transfection. We reserved the action time of tetracycline for 12h and the action time of rup2 for 7h, and observed the sealing of the sheet at 48h after transfection.

We observed the cell slides with different treatments under confocal microscope, and a field of view was found using a 20x magnification. Subsequently, find a field with more representative cells in different channels, and a multi-channel film was used to prepare for cell counting.

1. Wang Y, Wang L, Guan Z, et al. Structural insight into UV-B–activated UVR8 bound to COP1. Science Advances, 2022, 8(16): eabn3337.
2. Rizzini, L., Favory, J. J., Cloix, C., Faggionato, D., O’hara, A., Kaiserli, E., ... & Ulm, R. (2011). Perception of UV-B by the Arabidopsis UVR8 protein. Science, 332(6025), 103-106.
3. Jenkins, G. I. (2014). The UV-B photoreceptor UVR8: from structure to physiology. The Plant Cell, 26(1), 21-37.
4. Shimizu-Sato, S., Huq, E., Tepperman, J. M., & Quail, P. H. (2002). A light-switchable gene promoter system. Nature Biotechnology, 20(10), 1041-1044.
5. Yin, R., & Ulm, R. (2017). How plants cope with UV-B: from perception to response. Current Opinion in Plant Biology, 37, 42-48.