Part:BBa_K563004

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.

Characterization by [http://2016.igem.org/Team:Chalmers_Gothenburg iGEM Team Chalmers Gothenburg 2016]

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 OD=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=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.

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.

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.

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