Regulatory
pPYK2

Part:BBa_K2030003

Designed by: John Hellgren   Group: iGEM16_Chalmers_Gothenburg   (2016-08-17)
Revision as of 12:09, 19 October 2016 by Jannie (Talk | contribs)

pPYK2 S. cerevisiae promoter

The upstream regulatory sequence to the gene PYK2, coding for Pyruvate Kinase. Repressed by glucose [1].

Characterization

A promoter study was performed to characterize this promoter. The PYK2 promoter was cloned into the replicative plasmid p416tef by replacing the existing pTEF1 promoter and adding GFP as a reporter gene. By using a replicative plasmid instead of chromosomal integration, a higher copy number can be achieved, which will make sure that even weak promoters give a detectable signal. For the glucose conditions, the cells were grown as a preculture in SD -URA + 2 % glucose media overnight, diluted to OD600=0.3 in the same media and cultivated for 3 hours. The expression of GFP was measured in a 96-well plates (NUNC 96) in a BMG Labtech FLUOstar Omega plate reader with triplicate samples using the following setting: 20 flashes per well, excitation/emission wavelength at 485/520 nm and gain set to 800.

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. For the acetate experiment, the cells were grown as a preculture in SD -URA + 2 % glucose media overnight, washed and diluted to OD600=0.3 in SD -URA + 0.5 % acetate and cultivated for 24 hours before plate reader measurements. The longer cultivation time was due to slow growth with acetate as the carbon source. Furthermore, the reason for the longer cultivation time was to make sure that the GFP produced during the preculture in glucose was degraded.

The experiment was also done with the promoters pAQR1, pGLN1, pPCK1 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 sample, and the mean value of the negative control (cells with p416tef without GFP) was subtracted. The results are shown in Table 1.


Table 1. Fluorescent levels of GFP under the control of the promoters pAQR1, pGLN1, pPCK1,
PYK2 and pTEF1 for cells cultivated in SD -URA media + 2 % glucose or 0.5 % acetate (n=3).

Promoter Condition
Glucose (fluorescent unit/OD600)
 Acetate (fluorescent unit/OD600)
pAQR1
 303 63
pGLN1
 862 426
pPCK1 235 1721
pPYK2 125 77
pTEF1 1314 1399


In Figure 1 the results are normalized against the expression level of the pTEF1 promoter.


Figure 1: Fluorescent levels of GFP under the control of the promoters pAQR1, pGLN1, pPCK1, PYK2 and pTEF1 in glucose and acetate conditions relative the levels of pTEF1. Triplicate runs were made for each promoter and error bars are shown as confidence intervals with p=0.05, using student's t-test.

Uploads

Promoter study data

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]


References

[1] Boles E, Schulte F, Miosga T, Freidel K, Schlüter E, Zimmermann FK, et al. Characterization of a glucoserepressed pyruvate kinase (Pyk2p) in Saccharomyces cerevisiae that is catalytically insensitive to fructose-1,6-bisphosphate. Journal of Bacteriology. 1997;179(9):2987-93

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