Part:BBa_K4180008
pGal1,10-eGFP
This pGal1,10-eGFP 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. The enhanced GFP will be used as a control. It will be made into a composite part, pGal1, 10-eGFP-SBP (BBa_K4180008) as a control in the project.The galactose promoter can be induced in the presence of galactose and suppressed in the presence of glucose.
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. 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_K4180008 in BY4741 (use eGFP primers to do qPCR)>
Fig 3: BBa_K4180008 was used as a control for BBa_K4180005, BBa_K4180006, BBa_K4180007 composite parts transformed into BY4741 yeast strain, respectively. The purpose of doing timecourse in the presence of galactose was to determine whether our composite part could be induced by the galactose. In the presence of 2% YP-galactose for 17hr, eGFP was induced over 10-fold induction, even reached over 20-fold induction in the presence of 2% YP-galactose for 41hr, which indicated our team’s composite parts could be manipulated to dramatically induce the coding region downstream of Gal1,10 promoter in the presence of galactose.
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