Difference between revisions of "Part:BBa K3239010"

Revision as of 07:43, 16 October 2019

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 is activated, upregulating the expression of both the homogeneous Prm1 (which then self-upregulates itself) and the heterogeneous Mit1. This leads to a very strong activation of the AOX1 promoter.

Prm1 then activates pMIT1, upregulating the expression of the homogeneous Mit1. Unlike wildtype P. pastoris GS115 where Mit1 expression is then only upregulated by Prm1, in the constructed pAPM1-pAOX1-GFP strain, the heterogeneous Mit1 is also self-downregulated due to the downregulation of pPRM1 by Mit1. This corresponds to a much stronger activation pAOX1 compared to wildtype P. pastoris GS115 upon initial methanol induction. In later stages, pAOX1 activation might be weaker due to an accumulation of Mit1 that suppresses pPRM1. After Mit1 is fully degraded, however, pPRM1 inhibition shall be removed and pAOX1 shall be again strongly induced. Therefore the regulation of pAOX1 activity should be more like a cyclical process.

We included self-inhibition of Mit1 in the construct due to the fact that earlier research has demonstrated that if Mit1 is strongly self-upregulated by pAOX1 (which is activated by Mit1 and Prm1), the yeast cell 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, 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 pAPM1-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]

@@ Line 7: / Line 7: @@
 ===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.
+Upon methanol induction, pPRM1 is activated, upregulating the expression of both the homogeneous Prm1 (which then self-upregulates itself) and the heterogeneous Mit1. This leads to a very strong activation of the AOX1 promoter.
+Prm1 then activates pMIT1, upregulating the expression of the homogeneous Mit1. Unlike wildtype P. pastoris GS115 where Mit1 expression is then only upregulated by Prm1, in the constructed pAPM1-pAOX1-GFP strain, the heterogeneous Mit1 is also self-downregulated due to the downregulation of pPRM1 by Mit1. This corresponds to a much stronger activation pAOX1 compared to wildtype P. pastoris GS115 upon initial methanol induction. In later stages, pAOX1 activation might be weaker due to an accumulation of Mit1 that suppresses pPRM1. After Mit1 is fully degraded, however, pPRM1 inhibition shall be removed and pAOX1 shall be again strongly induced. Therefore the regulation of pAOX1 activity should be more like a cyclical process.
+We included self-inhibition of Mit1 in the construct due to the fact that earlier research has demonstrated that if Mit1 is strongly self-upregulated by pAOX1 (which is activated by Mit1 and Prm1), the yeast cell 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, 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.
-[[File:pGMP1-pAOX1-GFP.png|400px|thumb|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)]]
+[[File:pAPM1-pAOX1-GFP.png|400px|thumb|Figure 1. Illustration of the pAPM1-pAOX1-GFP construct (for in trans regulations among homogenous transcription factors and the homogenous AOX1, see the pAOX1-GFP construct)]]