Composite

Part:BBa_K3239010:Experience

Designed by: Xingyu Zhou   Group: iGEM19_ShanghaiFLS_China   (2019-10-16)
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Applications of BBa_K3239010

This construct is designed to moderately up-regulate the AOX1 promoter (PAOX1) activity in P. pastoris GS115. It is to be integrated into the PAOX1-GFP strain. Upon methanol induction, PPRM1 expression of the homogeneous PRM1 gene is activated, and further amplified via the self-upregulation of Prm1. Prm1 then activates PMIT1, upregulating the expression of the homogeneous MIT1 gene and the heterogeneous PRM1 gene. This overall leads to a strong activation of PAOX1 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-PAOX1-GFP strain, the heterogeneous MIT1 gene is also methanol induced as it is expressed by PPRM1. This corresponds to a much stronger activation PAOX1 compared to wild type 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 as earlier research has demonstrated that if Mit1 is strongly expressed and self-upregulated by PAOX1 (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 homogeneous AOX1 gene, the pAPM1-PAOX1-GFP strain should not only have a higher GFP yield compared to the PAOX1-GFP strain (see part BBa_K3239007) but also exhibit higher growth rates since it should be more capable of metabolizing methanol.

User Reviews

For detailed modeling, design, experiment, results, and demonstration, please check our wiki:

https://2019.igem.org/Team:ShanghaiFLS_China/Model

https://2019.igem.org/Team:ShanghaiFLS_China/Design

https://2019.igem.org/Team:ShanghaiFLS_China/Experiments

https://2019.igem.org/Team:ShanghaiFLS_China/Results

https://2019.igem.org/Team:ShanghaiFLS_China/Demonstrate


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