Difference between revisions of "Part:BBa K2631001"

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BD-ScFv2-pGAL1-eYFP

This construct base on the IGEM parts (BBa_K2247009) contains an anti-AFB1 single chain variable fragments (ScFv2) targeting different affinity sites of AFB1 were fused with DNA binding domain (BD) , and ADH1 terminator (transcription terminator for alcohol dehydrogenase 1). We put another ADH1 terminator, the GAL1 promoter (inducible promoter, regulated by Gal4 in yeast, is used as the promoter in a standard yeast-two-hybrid system) cloned from S. cerevisiae (Johnston M et al, 1984) and an eYFP (enhanced YFP mutated from Aequorea Victoria GFP protein) downstream from the ScFv2 in the BamHI site of the vector (Biteen J S et al, 2008). This part, together with AD-ScFv1 (BBa_K2247008), plays the central role in the AFB1 biosensor system. The presence of AFB1 would draw near the two ScFvs, thus enabling the association of Gal4 AD and BD domains, and activate the EYFP protein, and driving the expression of the reporter gene in the AH109 yeast strain (from Takara). This design is that expression of eYFP under the control of the GAL4 induced promoter it regulates would produce a feedback, making the construct more sensitive to the AFB1 in the environment. eYFP act as a signal of the system are working.

Sequence and Features

This scheme shows the working principle of our aflatoxin-b1 (AFB1) biosensor system. Two anti-AFB1 single chain variable fragments (ScFv) targeting different affinity sites of AFB1 were fused with Gal4 transcription activation domain (AD) and DNA binding domain (BD) respectively. In the presence of AFB1, the two anti-AFB1 ScFvs could be drawn near, enabling the association of Gal4 AD and BD, and thus driving the expression of genes under the Gal promoter. At the same time eYFP protein was also expressed under this condition.

The yeast cells that contain the two plasmids AD-ScFv1 plasmid and BD-ScFv2-pGAL1-eYFP were cultured in SD-His media with 100ug/L (100 ppb) and with the mock control. The result shows the eYFP is more enriched under the AFB1 (100ug/L) existing conditions in the yeast cells. According to our design, the presence of AFB1 should lead to the association of Gal4 AD and BD domains, and drive the expression of eYFP enabling the yeast show yellow fluorescence under the excitation light (expose time both at 200ms).

The yeast cells were cultured in SD-His media with 0ug/L, 100ug/L (100 ppb), 500ug/L(500 ppb), 1000ug/L(1000 ppb) Aflatoxin B1. According to our design, the presence of AFB1 should facilitate the association of Gal4 AD and BD, thus driving the expression of His3 and the eYFP protein under the Gal1 promoter, and enabling yeast growth in SD-His media and expressing reporter gene eYFP. Before loading samples all proteins were normalized by Bradford method. Results showed that, compared to control, the presence of AFB1 can induce the protein content of eYFP. Interestedly, under the content of 500ug/L AFB1 the eYFP shows significant increased band while under higher AFB1 at 1000ug/L there show more eYFP protein expression.

Reference：

Johnston M, Davis R W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.[J]. Molecular & Cellular Biology, 1984, 4(8):1440. Biteen J S, Thompson M A, Tselentis N K, et al. Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP.[J]. Nature Methods, 2008, 5(11):947-949.