Difference between revisions of "Part:BBa K801020"

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The characterization of this part was done using a KlADH4-promoter + eGFP construct.  
 
The characterization of this part was done using a KlADH4-promoter + eGFP construct.  
  
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In a first experiment, the transformed yeast cells were picked grown in a pre-culture (SC-U Medium) over night and transferred into SC-U Medium with different concentrations of ethanol (0%, 4%, 8%, 10%). The eGFP-fluorescence and the OD600 were measured at t = 0h, 3h, 18h, 21h, 24h.  
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In a first experiment, the transformed yeast cells were picked grown in a pre-culture (SC-U Medium, 30 °C, 180 rpm) over night and transferred into SC-U Medium with different concentrations of ethanol (0%, 4%, 8%, 10%). The eGFP-fluorescence and the OD600 were measured at t = 0h, 3h, 18h, 21h, 24h.  
  
 
For the evaluation of the experimental data, the measured fluorescence was divided by the respecitve OD600, to normalize the fluorescence to the respecitve cell count. This was done to take the intrinsic auto-fluorescence in account. The results are shown below:
 
For the evaluation of the experimental data, the measured fluorescence was divided by the respecitve OD600, to normalize the fluorescence to the respecitve cell count. This was done to take the intrinsic auto-fluorescence in account. The results are shown below:
  
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[[Image:TUM12_KlADH4_eGFP-expression_Graph1.jpg|600px]]
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[[Image:TUM12_KlADH4_eGFP-expression_Graph1.jpg|600px|thumb|center|Picture 1: First characterization experiment of the KlADH4-promoter (& eGFP) in ''S. cerevisiae''. After an overnight pre culture, the transformed yeast cells were transferred into SC-U Media with different ethanol concentration and the eGFP-fluorescence and the OD600 were measured at different times.]]
  
 
The promoter is generally functional in ''S. cerevisiae'', which can be seen by the fact that eGFP is expressed (also see  
 
The promoter is generally functional in ''S. cerevisiae'', which can be seen by the fact that eGFP is expressed (also see  

Revision as of 15:31, 24 September 2012

KlADH4 yeast promoter, ethanol inducible

This part is the ethanol inducible promoter controlling the KlADH4-gene of K. lactis.

The use of this ethanol inducible promoter to produce heterologous proteins in K. lactis was shown by Salioa et al. 1999 [http://www.ncbi.nlm.nih.gov/pubmed?term=9872759].

We characterized this part in S. cerevisiae (strain INVSc1) to find out whether this part is also ethanol-inducible in this yeast.


Usage and Biology

The UASe-region of this promoter has been shown to be responsible for the ethanol sensitivity of this promoter in K. lactis (Mazzoni et al., 2000 [http://www.ncbi.nlm.nih.gov/pubmed?term=10724480]). The region includes binding sites for the Rap1-protein (repressor activator protein 1) and the Yap1-protein (a transcription factor involved in stress response) as well as two heat shock elements (HSE) and five stress response elemtents (STRE). All these cis-elements and the respecitve proteins also occur in S. cerevisiae. For this reason we wanted to examine whether the KlADH4-promoter remains ethanol inducible if it is transferred from its natural organism (K. lactis) to S. cerevisiae.

The characterization of this part was done using a KlADH4-promoter + eGFP construct.

In a first experiment, the transformed yeast cells were picked grown in a pre-culture (SC-U Medium, 30 °C, 180 rpm) over night and transferred into SC-U Medium with different concentrations of ethanol (0%, 4%, 8%, 10%). The eGFP-fluorescence and the OD600 were measured at t = 0h, 3h, 18h, 21h, 24h.

For the evaluation of the experimental data, the measured fluorescence was divided by the respecitve OD600, to normalize the fluorescence to the respecitve cell count. This was done to take the intrinsic auto-fluorescence in account. The results are shown below:

Picture 1: First characterization experiment of the KlADH4-promoter (& eGFP) in S. cerevisiae. After an overnight pre culture, the transformed yeast cells were transferred into SC-U Media with different ethanol concentration and the eGFP-fluorescence and the OD600 were measured at different times.

The promoter is generally functional in S. cerevisiae, which can be seen by the fact that eGFP is expressed (also see

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 753
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 590


References

  • [1] Saliola, M., Mazzoni, C., Solimando, N., Crisà, A., Falcone, C. & Jung, G. (1999) ‚Use of the KlADH4 promoter for ethanol-dependent production of recombinant human serum albumin in Kluyveromyces lactis’, Appl Environ Microbiol. 65 (1), 53-60. [http://www.ncbi.nlm.nih.gov/pubmed?term=10724480 PMID: 9872759]
  • [2] Mazzoni, C., Santori, F., Saliola, M. & Falcone, C. (2000) ‚Molecular analysis of UASE, a cis element containing stress response elements responsible for ethanol induction of the KlADH4 gene of Kluyveromyces lactis’, Res. Microbiol. 151, 19-28. [http://www.ncbi.nlm.nih.gov/pubmed?term=10724480 PMID: 10724480]