Part:BBa_K4180005
pGal1,10-SNF1
This pGal1,10-SNF1 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.
Galactose promoter can be induced in the presence of galactose and express SNF1(FL) ; SNF1 in yeast is homology of AMPKα in human, which is a major metabolism sensor protein of the whole metabolic pathways, increasing catabolic process, such as triggering fatty acid catabolism for more ATP, and reducing anabolic process, such as protein and fatty acid synthesis to reduce energy consumed.
Citation: 1. Herzig, S. and R. J. Shaw (2018). "AMPK: guardian of metabolism and mitochondrial homeostasis." Nat Rev Mol Cell Biol 19(2): 121-135.
2. Yang, T T et al. “Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein.” The Journal of biological chemistry vol. 273,14 (1998): 8212-6. doi:10.1074/jbc.273.14.8212
Sequence and Features
<BBa_K4180005 in BY4741>
BBa_K4180005 composite site was SNF1 full-length coding region cloned downstream of the Gal1, 10 promoter. BBa_K4180005 composite site was transformed into BY4741 wild-type yeast strain. In the presence of 2%YP-galactose, SNF1 was only induced at least 3-fold at 17, and 24 hr, and 5-fold at 41 hr, which didn’t show if the BBa_K4180005 composite site was manipulated in the presence of galactose, compared to the induction in BBa_K4180008 control, since BBa_K4180005 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_K4180005 composite site was suppressed in the presence of galactose at 24hr.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1192
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 286
- 1000COMPATIBLE WITH RFC[1000]
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