Difference between revisions of "Team:ShanghaiFLS China/Design"

It has long been known that the AOX1 promoter (P_AOX1) is strongly induced by methanol and highly repressed by glucose and other carbon sources, yet it is only in the recent years that the in trans regulations are fully deciphered. As demonstrated in Wang et al., 2016, P_AOX1 is activated by a cascade of transcription factors Mxr1, Prm1, and Mit1: Mxr1 is essential for P_AOX1 de-repression and is inhibited by glucose. When methanol is present as the only carbon source, however, Mxr1 isderepressed, and Prm1 expression is induced by methanol. Prm1 expression is further amplified by its self-activation, while Mit1 expression is also upregulated by Prm1 activation. Taken together, Mxr1 derepresses P_AOX1, while Prm1 and Mit1 strongly activate P_AOX1, upregulating the expression of alcohol oxidase 1. Besides activating P_AOX1 though, Mit1 also represses the expression of Prm1, down regulating the cascade overall. The figure below illustrates this cascade upon methanol induction.

In trans regulation of P_AOX1 upon methanol induction in the wild type P. pastoris GS115 strain.

In order to further dynamically upregulate P_AOX1, we recombined the homologous promoters and transcription factors as our composite parts and integrated them into the wild type P. pastoris GS115 strain, which shall not only additionally activate P_AOX1 but also positively regulate the expression levels the homogeneous Mit1 and Prm1. We used yEGFP3 (abbr. GFP), a yeast optimized EGFP variant as our reporter gene of P_AOX1 activity.

Construct P_AOX1-GFP. The P_AOX1-GFP strain is obtained by integrating P_AOX1-GFP (Part:BBa_K3239007) into the wild type P. pastoris GS115. The GFP protein serves as the reporter of P_AOX1 activity. This serves as our control as P_AOX1 regulation is the same as the wild type P. pastoris GS115 strain.

Constructs pGMP1-P_AOX1-GFP, pAPM1-P_AOX1-GFP and pGMP1-pAPM1-P_AOX1-GFP. The pGMP1-P_AOX1-GFP strain is obtained by the integrating P_MIT1-PRM1 (Part:BBa_K3239009) into the P_AOX1-GFP strain; the pAPM1-P_AOX1-GFP strain is obtained by integrating P_PRM1-MIT1 (Part:BBa_K3239010) into the P_AOX1-GFP strain; and the pGMP1-pAPM1-P_AOX1-GFP is obtained by integrating P_MIT1-PRM1 and P_PRM1-MIT1 into the P_AOX1-GFP strain. In all of these strains, GFP expression reflects P_AOX1 activity. Note that while the illustration omits the homogenous AOX1 gene and the homogeneous in trans regulations among the homogeneous transcription factors, they are still present in the strains, and can be illustrated in the same way as in the figure above.

Overall design of the experiment. We first PCR amplified promoters P_PRM1 and P_MIT1 and genes PRM1 and MIT1 from pre-existing plasmids, then fused them together into plasmids pMIT1-PRM1 and pPRM1-MIT1 via Gibson assembly, hence leaving no scar at the conjunctoins. After PCR verifying each assembled plasmids, we then integrated them into P_AOX1-GFP strains via electroporation, giving us pGMP1-P_AOX1-GFP, pAPM1-P_AOX1-GFP and pGMP1-pAPM1-P_AOX1-GFP. Of our three constructs, two (pGMP1-P_AOX1-GFP, pAPM1-P_AOX1-GFP) were obtained and experimentally tested, while the third (pGMP1-pAPM1-P_AOX1-GFP) is obtained later and still being experimentally tested.

Given that this would eventually result in a very complicated regulatory cascade of P_AOX1 activation, we modeled our constructs for an approximate prediction of their expression efficiencies compared to wild type P. pastoris GS115. We later experimentally tested our constructs and gained fairly positive results.

Wang, X., Wang, Q., Wang, J., Zhou, M., Shi, L., Zhou, X., … Shen, W. (2016). Mit1 Transcription Factor Mediates Methanol Signaling and Regulates the Alcohol Oxidase 1 ( AOX1 ) Promoter in Pichia pastoris. Journal of Biological Chemistry, 291(12), 6245–6261. https://doi.org/10.1074/jbc.m115.692053