Difference between revisions of "Part:BBa K124002"

Revision as of 15:38, 10 October 2022

Yeast GPD (TDH3) Promoter

Regulatory region spanning 680 bp upstream of the start codon of the GPD1 gene in yeast.

Sequence and Features

Group: Tel-Hai 2017
Author: Yaakov Bulka
Summary: we added information about the promoter and on its mechanism.

This is a strong constitutive yeast expression promoter from glyceraldehyde 3-phosphage dehydrogenase. Also called TDH3 or GAPDH. Studies have found the promoter activity of TDH3 decreased significantly when glycerol or xylose was supplied as the carbon source and at high temperatures (42 °C) . Oxygen conditions had non-significant effect.

About the protein - the promoter was taken from the Glyceraldehyde-3-phosphate dehydrogenase peptide (GAPDH), that involves in glycolysis and gluconeogenesis. The protein is a tetramer that catalyzes the reaction of glyceraldehyde-3-phosphate to 1,3 bis-phosphoglycerate, detected in the cytoplasm and cell wall. GAPDH-derived antimicrobial peptides secreted by S. cerevisiae are active against a wide variety of wine-related yeasts and bacteria.

Partow, S., Siewers, V., Bjørn, S., Nielsen, J., & Maury, J. (2010). Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae. Yeast, 27(11), 955-964.‏

Yang, C., Hu, S., Zhu, S., Wang, D., Gao, X., & Hong, J. (2015). Characterizing yeast promoters used in Kluyveromyces marxianus. World Journal of Microbiology and Biotechnology, 31(10), 1641-1646.‏

Tianjin 2021's characterization

Group: Tianjin 2021
Author: Ruiqi Liu
Summary: we characterized TDH3 Promoter’s strength in 4742 yeast.

GFP is an important reporter gene used in our project. We integrated GFP gene into yeast’s chromosomes, and used it to represent the degradation of chromosomes. In order to verify whether the TDH3 promoter can express the GFP signal intensity required in experiment, we characterized the TDH3 promoter’s efficiency. We constructed a GFP fragment that was controlled by the TDH3 promoter and bound it to the chromosome genome of 4742 yeast. We measured the GFP expression levels of yeast at 15h, 20h,25h and 30h using microplate analyzer. Meanwhile, we used 4742 yeast (no GFP fragment) as control group.

Fig 1. Gene part using GFP as reporter gene to characterize TDH3 promoter strength

We use GFP as reporter gene to test the intensity of TDH3 promoter, and draw a bar chart with fluorescence intensity /OD as the ordinate and time as the abscissa. The excitation/emission wavelength of GFP in the microplate detector was set as 488/535nm.

Fig 2. Characterization of TDH3 promoter’s strength