Part:BBa_K4180006
pGal1,10-snf1Δ2-306aa
This pGal1,10-snf1Δ2-306aa is a composite biobrick. pGal1, 10-SPT5-Streptavidin Binding Protein(SBP) plasmid, containing 2u origin of replication (ORI) for yeast to start DNA replication, and ORI for bacteria DNA replication, was sponsored by Dr. Tien-Hsien Chang, at Genomics Research Center, Academia Sinica, in Taipei Taiwan. This plasmid can be transformed into bacteria and yeast, which was beneficial for our team to finish making biobricks in bacteria and perform the functional assay in yeast. Our team made 5 different basic parts (one promoter and 4 coding regions) and 4 different composite parts by inserting those 4 basic parts (BBa_K4180000, BBa_K4180002, BBa_K4180003, and BBa_K4180004) to replace the original SPT5 gene. pGal1,10-snf1Δ2-306aa, N-terminus truncated from amino acid 2 to 306 deleting kinase domain of SNF1, where the phosphorylation on Thr210 activates the catalytic activity of SNF1.Galactose promoter can be induced in the presence of galactose to drive the expression of the downstream coding gene, snf1Δ2-306aa.
Citation: 1. Investigations in Molecular Cell Biology (O'Connor); 13.1: Regulation of the GAL1 promoter; https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/Book%3A_Investigations_in_Molecular_Cell_Biology_(O'Connor)/13%3A_Protein_overexpression/13.01%3A_Regulation_of_the_GAL1_promoter
2. McCartney, R R, and M C Schmidt. “Regulation of Snf1 kinase. Activation requires phosphorylation of threonine 210 by an upstream kinase as well as a distinct step mediated by the Snf4 subunit.” The Journal of biological chemistry vol. 276,39 (2001): 36460-6. doi:10.1074/jbc.M104418200
Sequence and Features
<BBa_K4180006 in BY4741>
BBa_K4180006 composite site was SNF1 N-terminus truncated from amino acid 2 to 306 deleting kinase domain of SNF1, where the phosphorylation took place on Thr210 to activate the catalytic activity of SNF1, cloned downstream of the Gal1, 10 promoter. BBa_K4180006 composite site was transformed into BY4741 wild-type yeast strain. In the presence of 2%YP-galactose, SNF1 was only induced less than 2-fold at 24, and 41 hr, which didn’t show if the BBa_K4180006 composite site was manipulated in the presence of galactose, compared to the induction in BBa_K4180008 control, since BBa_K4180006 composite site was not manipulated as much as what our team expected compared to the control of the SNF1 induction in BBa_K4180008. Compared to the endogenous SNF1 inBY4741, the SNF1 induction in BBa_K4180006 composite site was suppressed in the presence of galactose.
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