Difference between revisions of "Part:BBa K124002"
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<i><h3>Background</h3></i>
 
<i><h3>Background</h3></i>
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<p>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. Firstly, we used glucose as the carbon source for enrichment, then used galactose as the carbon source for induction, and recorded the time of induction as zero point of time. The GFP expression levels of yeast at 15h, 20h,25h and 30h were measured by microplate analyzer. Meanwhile, we used 4742 yeast as control group.</p>
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<p>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.</p>
 
<i><h3>Result</h3></i>
 
<i><h3>Result</h3></i>
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<p>We tested the expression intensity of GFP by TDH3 promoter and expressed it by fluorescence intensity /OD600. The excitation/emission wavelength of GFP in the microplate detector was set as 488/535nm. The results clearly indicated that TDH1 promoter is not strong enough and thus not appropriate for the analysis of GFP in the 4742 yeasts.</p>
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<p>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.</p>
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[[File:T--Tianjin--Parts3.png|500px|thumb|left|Figure 1.Result]]
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[[File:T--Tianjin--Parts3.png|500px|thumb|left|<i>Fig 2. Characterization of TDH3 promoter’s strength</i>]]
  
 
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<!-- Uncomment this to enable Functional Parameter display  

Revision as of 05:27, 21 October 2021

Yeast GPD (TDH3) Promoter

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

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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

Characterization of TDH3 Promoter’s GFP expression in 4742 yeast

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

Background

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.

Result

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.

Fig 2. Characterization of TDH3 promoter’s strength