In our project, we not only added some new parts and new information, including experimental data to the Part Register, but also provided protocols and other reference information for yeast two-hybrid experiments. Last but not least, we hope our solution can contribute to the more wildly implementation of CAR-T to treat tumors.

 

	Part Contributions
Part Number	Part Name	Contribution Type
BBa_K4904003	AD-SPA1-N545	New part
BBa_K4904002	BD-CRY2-N489-GFP	New part

 

	Other Contributions
Name	Description	Contribution Type
Optimization of CAR-T	In the modification of the CAR gene, a blue light control switch was added to the expression of the CAR gene to control its expression.	Optimization of existing treatments

 

The yeast two-hybrid technique was initially established by Fields et al[1]. when studying the properties of yeast transcription factor GAL4 and has been developed into a mature protein-protein interaction research technique after continuous improvement. It is widely used in the screening of interacting proteins, the identification and verification of protein interactions, the exploration of protein interaction mechanisms, and the mapping of protein linkage.

 

Transcription of yeast initiation genes requires the involvement of trans-transcriptional activators. Trans transcriptional activators, in our experiment, GAL4, include two separate but functionally essential domains: DNA binding domain (DNA-BD) located at N-terminal amino acid residues at positions 1-147 and Activation environment (AD) located at C-terminal amino acid residues at positions 768-881, respectively. DNA-BD recognizes and binds to the Upstream activating sequence (UAS) of Gal4-activated genes. On the other hand, AD initiates the downstream genes of UAS for transcription by binding with other components of the transcription machinery. DNA-BD and AD act separately and cannot activate the transcription reaction. However, when the two are close enough in space, it showed complete GAL4 transcription factor activity. It activated the downstream promoter of UAS so that the downstream genes of the champion could be transcribed.

 

 

The blue-light-activated CRY2 protein forms photobodies through phase separation to recruit the m6A "encoder" complex, which regulates transcriptome methylation. m6A methylation changes mRNA's degradation rate, the biological clock's core component, thus affecting the circadian clock's light response. [2]

Green fluorescent protein (GFP) is a β-barrel-shaped protein 1 composed of 238 amino acids with a molecular weight of about 27 kDa. GFP is derived from the crystal jellyfish Aequorea victoria. GFP can transform the blue fluorescence emitted by the jellyfish luminescent protein through chemical interaction into green fluorescence through energy transfer.[3] GFP has an excitation wavelength of 488 nm and an emission peak of about 507 nm.In this project, the effectiveness of the switch is tested by constructing a GFP reporting system.

In regard to this part, we conducted the plasmid construction, electrophoresis gel validation, sequencing, blue light reactivity verification and GFP fluorescence expression detection and all the data was well documented for future igmers’ use.

 

 

SPA1 is one of the three major negative regulators of Arabidopsis photomorphogenesis.[4] This project will compare the sensitivity of existing mature CRY2/CIB1 switches. The preliminary test results show that CRY2/SPA1 will be more sensitive than CRY2/CIB1. This is the main innovation of this project. In addition to the experimental date from the plasmid construction, we also obtained the blue light reactivity verification and sensitivity evaluation compared to CRY2/CIB1. Especially, we conducted β-Galactose activity test for  Gal4-activated gene in AD-SPA1-N545 where the β-Galactose activity indicates an increasing trend through time

 

Cart through genetic engineering technology, T cells are activated and equipped with a positioning and navigation device CAR (tumor chimeric antigen receptor), the ordinary "soldier" of T cells is transformed into a "super soldier," that is, CAR T cells, using its "positioning and navigation device" CAR, specifically identify tumor cells in the body, and release a large number of various effect factors through immune action. They can effectively kill tumor cells to achieve the purpose of treating malignant tumors.

 

The development cost of CAR-T is high, mainly due to some expensive production materials—for example, lentiviral vectors are used for transduction, and magnetic beads are used for cell sorting.

This technology is used to modify the CAR gene to optimize it by adding a blue light-regulated switch to the expression of the CAR gene to control its expression. Since if you don't own it, CAR will keep expressing itself. CAR targets sites on cancer cells and normal cells, but to a lesser extent. But it's still a thousand kills and three thousand kills, so the side effects are enormous. Through the optimization of the blue light control switch, specific and strictly controllable immunotherapy response to tumor cells can be achieved under the dual activation conditions of tumor antigen and light.

 

1) Fields S, Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989;340:245–246.

2) Xu W,Bochen J,Lianfeng G, et al. A photoregulatory mechanism of the circadian clock in Arabidopsis[J]. Nature Plants,2021,7(10).

3) Tsien, R. Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

4) Liu, B., Zuo, Z., Liu, H., Liu, X. & Lin, C. Arabidopsis cryptochrome one interacts with SPA1 to suppress COP1 activity in response to blue light—genes Dev. 25, 1029–1034 (2011).