Difference between revisions of "Part:BBa K801020"

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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 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''.
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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.
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 15:02, 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) 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.

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]