Difference between revisions of "Part:BBa K2030001"
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===Characterization===
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==Contribution ==
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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. 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.
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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 co-culture 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.  
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*'''Group:''' [http://2016.igem.org/Team:Chalmers_Gothenburg iGEM Team Chalmers Gothenburg 2016]
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*'''Author:''' John Hellgren
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*'''Summary:''' A promoter study to characterize this promoter and compare it against several others in two different conditions.
  
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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 OD<SUB>600</SUB> of that sample, and the mean value of the negative control (cells with p416tef without GFP) was subtracted. The results are shown in <b>Figure 1</b>. In <b>Figure 2</b> the results are normalized against the expression level of the pTEF1 promoter.
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===Characterization ===
 +
A promoter study was performed to characterize this promoter. The reporter gene GFP was cloned into the replicative plasmid p416tef, downstream of the <i>TEF1</i> promoter. 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.
 +
 
 +
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.
 +
 
 +
The experiment was also done with the promoters pAQR1, pGLN1, ,pPCK1 and pPYK2 in the same way, and the results compared against each other. The raw data from the promoter study was normalized against OD<SUB>600</SUB> of that sample, and the mean value of the negative control (cells with p416tef without GFP) was subtracted. The results are shown in <b>Figure 1</b>. In <b>Figure 2</b> the results are normalized against the expression level of the pTEF1 promoter.
  
 
<p>[[File:T--Chalmers Gothenburg--glucose-acetate.png]]
 
<p>[[File:T--Chalmers Gothenburg--glucose-acetate.png]]
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<br><b>Figure 1. Expression levels of the promoters pAQR1, pGLN1, pPCK1, PYK2 and pTEF1 in glucose and acetate conditions. Error bar are shown as confidence intervals with p=0.05, using students t-test. </b></p>
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<br><b>Figure 1. Expression levels of the promoters pAQR1, pGLN1, pPCK1, PYK2 and pTEF1 in glucose and acetate conditions. Triplicate runs were made for each promoter and error bar are shown as confidence intervals with p=0.05, using students t-test. </b></p>
  
 
<p>[[File:T--Chalmers Gothenburg--glucose-acetate-relative.png]]
 
<p>[[File:T--Chalmers Gothenburg--glucose-acetate-relative.png]]
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<br><b>Figure 2. Expression levels of the promoters pAQR1, pGLN1, pPCK1, PYK2 and pTEF1 in glucose and acetate conditions relative the levels of pTEF1. Error bar are shown as confidence intervals with p=0.05, using students t-test. </b></p>
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<br><b>Figure 2. Expression levels 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 bar are shown as confidence intervals with p=0.05, using students t-test. </b></p>
  
 
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Revision as of 08:49, 14 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].


Contribution

  • Group: [http://2016.igem.org/Team:Chalmers_Gothenburg iGEM Team Chalmers Gothenburg 2016]
  • Author: John Hellgren
  • Summary: A promoter study to characterize this promoter and compare it against several others in two different conditions.

Characterization

A promoter study was performed to characterize this promoter. The reporter gene GFP was cloned into the replicative plasmid p416tef, downstream of the TEF1 promoter. 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 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. 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 pPYK2 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 Figure 1. In Figure 2 the results are normalized against the expression level of the pTEF1 promoter.

T--Chalmers Gothenburg--glucose-acetate.png
Figure 1. Expression levels of the promoters pAQR1, pGLN1, pPCK1, PYK2 and pTEF1 in glucose and acetate conditions. Triplicate runs were made for each promoter and error bar are shown as confidence intervals with p=0.05, using students t-test.

T--Chalmers Gothenburg--glucose-acetate-relative.png
Figure 2. Expression levels 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 bar are shown as confidence intervals with p=0.05, using students t-test.

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.