Difference between revisions of "Part:BBa K3239010"

Latest revision as of 06:57, 18 October 2019

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]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3239010 short</partinfo>
-MIT1 gene expressed by the pPRM1 promoter.
+''MIT1'' gene expressed by the ''P<sub>PRM1'' promoter.
 ===Usage and Biology===
-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.
+Upon methanol induction, ''P<sub>PRM1'' expression of the homogeneous ''PRM1'' gene is activated, and further amplified via the self-upregulation of Prm1. Prm1 then activates ''P<sub>MIT1'', upregulating the expression of the homogeneous ''MIT1'' gene and the heterogeneous ''PRM1'' gene. This overall leads to a strong activation of ''P<sub>AOX1'' expression of the ''AOX1'' gene.
-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.
+Unlike in wild type ''P. pastoris GS115'' where ''MIT1'' gene expression is then only upregulated by Prm1, in the constructed ''pAPM1''-''P<sub>AOX1''-GFP strain, the heterogeneous ''MIT1'' gene is also methanol induced as it is expressed by ''P<sub>PRM1''. This corresponds to a much stronger activation ''P<sub>AOX1'' compared to wild type ''P. pastoris GS115'' upon initial methanol induction. In later stages, ''P<sub>AOX1'' activation might be weaker due to an accumulation of Mit1 that suppresses ''P<sub>PRM1''. After Mit1 is fully degraded, however, ''P<sub>PRM1'' inhibition shall be removed and ''P<sub>AOX1'' shall be again strongly induced. Therefore the regulation of ''P<sub>AOX1'' 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.
+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<sub>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, 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.
+Given that in constructed strains, ''P<sub>AOX1'' not only expresses the ''yEGFP3'' reporter gene, but also the homogeneous ''AOX1'' gene, the ''pAPM1''-''P<sub>AOX1''-GFP strain should not only have a higher GFP yield compared to the ''P<sub>AOX1''-GFP strain (see part BBa_K3239007) but also exhibit higher growth rates since it should be more capable of metabolizing methanol.
-[[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)]]
+[[File:pAPM1-pAOX1-GFP.png|400px|thumb|Figure 1. Illustration of the ''pAPM1''-''P<sub>AOX1''-GFP construct (for ''in trans'' regulations among homogeneous transcription factors and the homogeneous ''AOX1'', see the ''P<sub>AOX1''-GFP construct in part BBa_K3239007)]]