Difference between revisions of "Part:BBa K2030001"

Revision as of 09:53, 13 October 2016

pPCK1 S. cerevisiae promoter

The upstream regulatory sequence to the gene PCK1, coding for Phosphoenolpyruvate carboxykinase. This promoter is induced by glycerol, acetate and ethanol (10x) [1].

Characterization

A promoter study was performed to characterize this promoter. The PCK1 promoter cloned into the replicative plasmid p416tef by replacing the existing pTEF1 promoter and adding GFP as a reporter gene. For the glucose conditions, the cells were grown in SD -URA + 2 % glucose media. The expression of GFP was measured using a plate reader with triplicate samples. The cells were also grown in SD -URA + 0.5 % acetate to compare the expression levels when acetate was the only carbon source, which is connected to our coculture project.

The experiment was also done with the promoters pAQR1, pGLN1, pPYCK2 and pTEF1 in the same way, and the results compared against each other. The raw data from the promoter study was normalized against OD600 of that value, and the mean value of the negative control (cells with p416tef without GFP) was subtracted. In figure 2, the data is normalized against the expression of the pTEF1 promoter.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 221
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 364
1000
COMPATIBLE WITH RFC[1000]

References

[1] K. Weinhandl, M. Winkler, A. Glieder, and A. Camattari, “Carbon source dependent promoters in yeasts,” Microbial Cell Factories, vol. 13, no. 1, 2014.

@@ Line 7: / Line 7: @@
 ===Characterization===
+A promoter study was performed to characterize this promoter. The PCK1 promoter cloned into the replicative plasmid p416tef by replacing the existing pTEF1 promoter and adding GFP as a reporter gene. For the glucose conditions, the cells were grown in SD -URA + 2 % glucose media. The expression of GFP was measured using a plate reader with triplicate samples. The cells were also grown in SD -URA + 0.5 % acetate to compare the expression levels when acetate was the only carbon source, which is connected to our coculture project.
+The experiment was also done with the promoters pAQR1, pGLN1, pPYCK2 and pTEF1 in the same way, and the results compared against each other. The raw data from the promoter study was normalized against OD600 of that value, and the mean value of the negative control (cells with p416tef without GFP) was subtracted.  In figure 2, the data is normalized against the expression of the pTEF1 promoter.
 [[File:T--Chalmers Gothenburg--glucose-acetate.png]]