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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 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 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> | + | 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>Table 1</b>. In <b>Figure 1</b> the results are normalized against the expression level of the pTEF1 promoter. |
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| + | <br><b>Table 1. Expression levels of the promoters pAQR1, pGLN1, pPCK1, PYK2 and <br> | ||
| + | pTEF1 in glucose and acetate conditions relative the levels of pTEF1.</b> | ||
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<p>[[File:T--Chalmers Gothenburg--glucose-acetate-relative.png]] | <p>[[File:T--Chalmers Gothenburg--glucose-acetate-relative.png]] | ||
| − | <br><b>Figure | + | <br><b>Figure 1. Expression levels of the promoters pAQR1, pGLN1, pPCK1, PYK2 and pTEF1 in glucose and acetate conditions relative the levels of pTEF1. Error bars are shown as confidence intervals with p=0.05, using student's t-test. </b></p> |
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Revision as of 09:15, 14 October 2016
yeast TEF1 promoter
This is the full length TEF1 promoter including the distal and proximal portions and their regulatory elements.
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 Table 1. In Figure 1 the results are normalized against the expression level of the pTEF1 promoter.
Table 1. Expression levels of the promoters pAQR1, pGLN1, pPCK1, PYK2 and
pTEF1 in glucose and acetate conditions relative the levels of pTEF1.
| 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 |
Figure 1. Expression levels of the promoters pAQR1, pGLN1, pPCK1, PYK2 and pTEF1 in glucose and acetate conditions relative the levels of pTEF1. Error bars are shown as confidence intervals with p=0.05, using student's t-test.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 205