Part:BBa_K4211054
LightOn-CenA
LightOn system is a blue light-activated system. The research team from East China University of Science and Technology developed a simple robust transgene system that is directly regulated by a single genetically encoded, photosensitive transactivator in 2012. The system can rapidly bind the positive regulator Gal4(65) with 5×UAS(G) under illumination with blue light.
Based on the article, The BNUZH-China iGEM team 2022 designed a blue light-activated system. We fused 5×UAS(G) (BBa_K4211005) with TATA box (BBa_K4211029). Therefore, we fused Pre LightOn promoter (BBa_K4211026) in front of the 5×UAS(G) and Post LightOn promoter (BBa_K4211030) behind the TATA box. Then we linked the gene of interest. Therefore, we added a TAA (BBa_M36117) sequence to terminated the transcription of the gene of interest. At the C-terminal of the gene of interest, we added a pCMV promoter (BBa_K2364000) to control the expression of the downstream target genes. In order to sense the blue light, we bound Gal4(65) (BBa_K4211019), VVD (BBa_K4211021) and p65 Activation Domain (BBa_K4211023) at the C-terminal of the pCMV promoter (BBa_K2364000). The Gal4 transcription factor is a positive regulator of gene expression of galactose-induced genes. This protein represents a large fungal family of transcription factors, Gal4 family, which includes over 50 members in the yeast Saccharomyces cerevisiae. Gal4(65) is a 65-amino acid residue in Gal4 protein (Gal4 residue 1-65). VVD, the smallest light-oxygen-voltage (LOV) domain–containing protein, forms a rapidly exchanging dimer upon blue-light activation. The correctly folded VVD domain in the fusion protein can be bound by flavin adenine dinucleotide (FAD). p65 Activation Domain, known as GAVP, is a transactivator. After light activation, GAVP homodimerizes, interacts with UASG elements (5×UASG) and initiates expression of the gene of interest. When illuminated with the blue light, Gal4(65)-VVD can bound the 5×UAS(G) sequence upon blue-light illumination. After that, 2 Gal4(65)-VVD will combine with each other, activating the transcription of the downstream target gene.
Bacterium Cellulomonas fimi uses 3 endoglucanases and an exoglucanase in the degradation of cellulose into cellobiose, before using β-glucosidase to catalyse the conversion of cellobiose to D-glucose. CenA is used to cut internal β-1,4-glucosidic bonds.
In this module, we replaced GOI with IL-2-CenA. We hoped that when illuminated with the blue light, CenA can be transcription and translation. Then is secreted into the extracellular and works, cutting internal β-1,4-glucosidic bonds. Figure 1 illustrates the detailed design of the whole device.
Figure 1: Construct design and the principle of the LightOn-CenA. 5×UAS(G) was fused to the TATA box and IL-2-CenA. Gal4(G) was linked via a linker to VVD. At the same time, VVD was combined with p65 Activation Domain though a linker. When the blue light illuminating, Gal4(65) combines with 5×UAS(G). Then, 2 Gal4(65)-VVD will combine with each other, activating the transcription of the downstream target gene: IL-2-CenA. Then CenA is secreted into the extracellular and works, cutting internal β-1,4-glucosidic bonds.
Usage and Biology
We used it to activate the transcription of CenA (BBa_K4211017) to cut internal β-1,4-glucosidic bonds.
Experimental approach
For testing this device, we used HEK293T cells, which were seeded in 25cm2 flask. The PLVX vector was transiently transfected into HEK293T cells by Lipo8000™ transfection system and incubated for 48 h to ensure normal cell status. Then, the system was illuminated with blue light (460 nm) for 3 h. After illumination, the cells were observed and the culture medium supernatant was analyzed by SDS-PAGE and Western Blot.
Figure 2: Western blotting analysis of CenA. CenA molecular weight is about 48 kDa.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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