1. Background information

Optogenetics initially emerged as a tool for light-inducible activation or suppression of membrane channel currents in the neurons of various subtypes. By combining light-sensing module and transcription factor module optogenetic tools can be used to regulate gene transcription in synthetic biology, targeted therapy of diseases and other fields.(Lan et al.; Liu et al.)Compared with chemical drug induction, the light-controlled optogenetic switches has several advantages : not requir ed ch emical compound absorption process and much faster; e asy for automation of production; small toxic effect; more convenient to switch on and off and so on. In the field of medicine, there is a significant need for precise drug dosing, localized drug delivery, and timed drug administration . As an important application prospect, optogenetic switches hold great promise in targeted therapy for major diseases such as cancer, diabetes, and neurological disorders.

Currently, the most commonly used optogenetic switches are blue light optogenetic switch and red light/far-redoptogenetic switch.The chromophore phytochromobilin (PΦB) is only found in blue-green algae and plants, limiting the applicability of red light/far-red light control switches. Blue light is the most promising development in optogenetic switch systems. Among blue light systems, CRY2/CIB1 has been the most extensively researched. Its "on" process of blue light optogenetic switchis controlled by blue light irradiation, while the "off" process requires CRY2, which is activated, to gradually degrade or gradually return to the excited state after being in the dark.

However, all blue light switch systems, including CRY2/CIB1, and even ultraviolet light control systems, lack an active control process for "off" like in red light/far-red light control switches. Instead, they rely on passive processes, where the switch components gradually return to a non-activated state or degrade when placed in darkness(Q. Wang et al.). Therefore, the mainstream optogenetic switch system, such as CRY2/CIB1, is in need of improvement.

BIC1 and BIC2 were discovered in 2016 as important components of the plant blue light signaling pathway ( Wang, Q., et al. ) . They have the ability to revert the blue light-activated CRY1/CRY2 to their non-activated state and can interact with light-activated CRY, CIB1, thereby shutting down the blue light signaling pathway. The complex structure of BIC2 with CRY2 and the dimeric structure of UVR8 have been elucidated. The N-terminus of UVR8 can sense ultraviolet (UVB) light signals and dissociate into monomers. In this project, we will leverage the unique characteristics of both to construct a UVR8N-BIC2 fusion protein to optimize the "off" operation of the blue light-dependent optogeneticswitch system CRY2/CIB1, allowing it to quickly complete the shutdown under UVB irradiation.

Figure 1 . General concept of our project.

As shown in Figure1: w hen exposed to blue light with a wavelength of 400-500nm, the receptor CRY2 is activated by the blue light. BD-CRY2 binds to AD-CIB1, initiating the transcription of downstream genes under the Gal4 UAS promoter. When exposed to 311nm UVB light, the UVR8-BIC2 fusion protein dissociates into monomers. BD-CRY2 competes with UVR8-BIC2 for binding, causing CRY2 to transition into a non-blue light-activated state, leading to a rapid cessation of transcription of downstream genes. In contrast, in the absence of UVB, the original system requires the slow recovery of light-activated CRY2 to its non-activated state or its degradation to shut down transcription of downstream genes. Therefore, this system has a more efficient "off" process.

2. Design

First of all, we will design primers and amplify TRP promoter fragment from pBridge vector, UVR8N397 fragment from the plasmid containing UVR8 CDS sequence, and BIC2 fragment from Arabidopsis thaliana genome cDNA by PCR assay. We will also construct linearized pBridge-BD-CRY2 vector by enzyme digestion. Then we will perform homologous recombination to link the 3 DNA fragments to the linear pBridge-BD-CRY2 vector. And we will get the plasmid pBridge-BD-CRY2(UVR8-BIC2).

Next we will transform the recombinant plasmid into E.coli.After this step, a massive number of the plasmid could be extracted from the bacteria. A small amount of this sample will be sent to genetic sequencing agencies to test if the construction of the plasmid has been successful. Then, we will transform the plasmid pBridge-BD-CRY2(UVR8-BIC2) and AD-CIB1 into yeast AH109. After this, we will perform β-galactosidase activity assay to detect the feasibility of the system .

3. Goal

a. The plasmid pBridge-BD-CRY2(UVR8-BIC2) is successfully constructed, which is identified by enzyme single digestion and sequencing.

b. UVR8-BIC2 protein products with biological functions could be obtained from E. coliand AH109. β-galactosidase activity assay show that UVR8N-BIC2 fusion protein that responds to UVB depolymerization to optimize the process of CRY2/CIB1 off .

c. In addition, we hope that the research results of this topic have broad application prospects. We hope we can use this research to improve the speed of light switch "off", so as to benefit people with diabetes or other diseases.

4. References and sources

Cloix, C., et al. "C-Terminal Region of the Uv-B Photoreceptor Uvr8 Initiates Signaling through Interaction with the Cop1 Protein." Proceedings of the National Academy of Sciences of the United States of America 109.40 (2012): 16366-70. Print.

Lan, T. H., et al. "Optogenetics for Transcriptional Programming and Genetic Engineering." Trends Genet 38.12 (2022): 1253-70. Print.

Liu, H., et al. "Optogenetic Control of Transcription in Zebrafish." PLoS One 7.11 (2012): e50738. Print.

Rizzini, L., et al. "Perception of Uv-B by the Arabidopsis Uvr8 Protein." Science 332.6025 (2011): 103-6. Print.

Wang, Q., et al. "Photoactivation and Inactivation of Arabidopsis Cryptochrome 2." Science 354.6310 (2016): 343-47. Print.

Wang, X., et al. "A Cry-Bic Negative-Feedback Circuitry Regulating Blue Light Sensitivity of Arabidopsis." Plant J 92.3 (2017): 426-36. Print.