Part:BBa_K3239010

pPRM1-MIT1

MIT1 gene expressed by the P_PRM1 promoter.

Usage and Biology

Upon methanol induction, P_PRM1 expression of the homogeneous PRM1 gene is activated, and further amplified via the self-upregulation of Prm1. Prm1 then activates P_MIT1, upregulating the expression of the homogeneous MIT1 gene and the heterogeneous PRM1 gene. This overall leads to a strong activation of P_AOX1 expression of the AOX1 gene.

Unlike in wild type P. pastoris GS115 where MIT1 gene expression is then only upregulated by Prm1, in the constructed pAPM1-P_AOX1-GFP strain, the heterogeneous MIT1 gene is also methanol induced as it is expressed by P_PRM1. This corresponds to a much stronger activation P_AOX1 compared to wild type P. pastoris GS115 upon initial methanol induction. In later stages, P_AOX1 activation might be weaker due to an accumulation of Mit1 that suppresses P_PRM1. After Mit1 is fully degraded, however, P_PRM1 inhibition shall be removed and P_AOX1 shall be again strongly induced. Therefore the regulation of P_AOX1 activity should be more like a cyclical process.

We included self-inhibition of Mit1 in the construct as earlier research has demonstrated that if Mit1 is strongly expressed and self-upregulated by P_AOX1 (which is activated by Mit1 and Prm1), the yeast will not survive. This suggests that Mit1 is slightly cytotoxic and its expression should be moderated at an appropriate level.

Given that in constructed strains, P_AOX1 not only expresses the yEGFP3 reporter gene, but also the homogeneous AOX1 gene, the pAPM1-P_AOX1-GFP strain should not only have a higher GFP yield compared to the P_AOX1-GFP strain (see part BBa_K3239007) but also exhibit higher growth rates since it should be more capable of metabolizing methanol.

Figure 1. Illustration of the pAPM1-P_AOX1-GFP construct (for in trans regulations among homogeneous transcription factors and the homogeneous AOX1, see the P_AOX1-GFP construct in part BBa_K3239007)

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]