Part:BBa_K3239010

pPRM1-MIT1

MIT1 gene expressed by the pPRM1 promoter.

Usage and Biology

This construct is designed to moderately up-regulate the AOX1 promoter (pAOX1) activity in P. pastoris GS115. It is to be homologously recombined into the pAOX1-GFP strain. Upon methanol induction, pPRM1 expression of the homogeneous Prm1 is activated, and further amplified via the self-upregulation of Prm1. Prm1 then activates pMIT1, upregulating the expression of the homogeneous Mit1 and the heterogeneous Prm1. Unlike wildtype P. pastoris GS115 where Prm1 expression is then inhibited by Mit1, in the constructed pGMP1-pAOX1-GFP strain, the heterogeneous Prm1 is constantly self-upregulated due to the upregulation of pMIT1 by Prm1. This leads to an overall upregulation of the expression of pAOX1 transcription factors and hence shall up-regulate pAOX1 activity compared to wildtype P. pastoris. Given that in constructed strains, pAOX1 not only expresses the yEGFP3 reporter gene, but also the homogenous AOX1 (alcohol oxidase 1) gene, the pGMP1-pAOX1-GFP strain should not only have a higher GFP yield compared to the pAOX1-GFP strain (pAOX1-GFP homologously recombined into wildtype P. pastoris GS115) but also exhibit higher growth rates since it should be more capable of metabolizing methanol.

Figure 1. Illustration of the pGMP1-pAOX1-GFP construct (for in trans regulations among homogenous transcription factors and the homogenous AOX1, see the pAOX1-GFP construct)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 2995
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 2995
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 293
  Illegal BglII site found at 3453
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 2995
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 2995
  
• 1000
  COMPATIBLE WITH RFC[1000]