Difference between revisions of "Part:BBa K563004"

Revision as of 09:50, 15 October 2016

pTEF1, promoter of Translational elongation factor EF-1 alpha

TEF1 encodes Translational elongation factor EF-1 alpha. The transcription level of the promoter is very high all the time. In our project, we constrct pTEF1-Tor, to overexpress Tor.

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. 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 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 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
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 205

@@ Line 12: / Line 12: @@
 ===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.
+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. 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.
 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.
@@ Line 18: / Line 18: @@
 The experiment was also done with the promoters [https://parts.igem.org/Part:BBa_K2030000 pAQR1], [https://parts.igem.org/Part:BBa_K2030004 pGLN1], [https://parts.igem.org/Part:BBa_K2030001 pPCK1] and [https://parts.igem.org/Part:BBa_K319003 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>Table 1</b>.
-<br><b>Table 1. Fluorescent levels of GFP under the control of the promoters pAQR1, pGLN1, pPCK1, PYK2 and <br>
+<br><center><b>Table 1. Fluorescent levels of GFP under the control of the promoters pAQR1, pGLN1, pPCK1, <br>PYK2 and pTEF1 for cells cultivated in SD -URA media + 2 % glucose or 0.5 % acetate (n=3).</b>
-pTEF1 in glucose and acetate conditions (n=3).</b>
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+</center>
 <br>
 <p>In <b>Figure 1</b> the results are normalized against the expression level of the pTEF1 promoter.</p>
-[[File:T--Chalmers Gothenburg--glucose-acetate-relative.png]]
-<br><b>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. </b>
+[[Image:T--Chalmers Gothenburg--glucose-acetate-relative.png|800px|thumb|center|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.]]