Difference between revisions of "Part:BBa K2030003"

Latest revision as of 16:56, 19 October 2016

pPYK2 S. cerevisiae promoter

The upstream regulatory sequence to the gene PYK2 from Saccharomyces cerevisiae CEN.PK113-5D, 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.

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. Each sample was loaded into three different wells in the plate reader, and error bars are shown as confidence intervals with p = 0.05, using student's t-test.

All promoters except pPCK1 show higher expression relative pTEF1 at glucose conditions compared with acetate conditions, which is consistent with previous reports [2]. pPCK1 even has higher expression level than pTEF1, which means that pPCK1 could be preferred for overexpression when acetate is the only carbon source.

A more detailed version of the promoter study and how it's connected to our project can be found [http://2016.igem.org/Team:Chalmers_Gothenburg/Project/Promoter_study here].

Uploads

Promoter study data

Sequence and Features

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