Difference between revisions of "Part:BBa K2030001"

Revision as of 12:22, 16 October 2016

pPCK1 S. cerevisiae promoter

The upstream regulatory sequence to the gene PCK1 from Saccharomyces cerevisiae, 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 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 using a BMG Labtech FLUOstar Omega plate reader with excitation/emission wavelength set at 485/520 nm, using 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. 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, pPYK2 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
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 5: / Line 5: @@
 ===Characterization===
-A promoter study was performed to characterize this promoter. The <i>PCK1</i> 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 OD<SUB>600</SUB>=0.3 in the same media and cultivated for 3 hours. The expression of GFP was measured using a plate reader with triplicate samples.
+A promoter study was performed to characterize this promoter. The <i>PCK1</i> 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 OD<SUB>600</SUB>=0.3 in the same media and cultivated for 3 hours. The expression of GFP was measured using a BMG Labtech FLUOstar Omega plate reader with excitation/emission wavelength set at 485/520 nm, using 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. For the acetate experiment, the cells were grown as a  preculture in SD -URA + 2 % glucose media overnight, washed and diluted to OD<SUB>600</SUB>=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.