OVERVIEW

​ Hoping to make a useful contribution for future iGEM teams, we completed the experimental characterization of an existing part Ethanol regulated promoter AOX1(BBa_I764001) and provided new data for it.

Biobricks	codes in the lab	Quantitative Characterization
BBa_I764001	promoter	fluorescence intensity

The details are in the part registrations page.

CONTRIBUTION

P_AOX1

​ The AOX1 promoter（P_AOX1） region from Pichia pastoris. It was first registered in 2007 and used as a strong promoter in Pichia pastoris. A complex pathway for the metabolism of methanol exists within some species of the Komagataella genus. Alcohol oxidase (AO) appears to be the first and major enzyme produced in this metabolic pathway. Transcribed from its gene (AOX1), AO converts methanol to formaldehyde within the yeast's peroxisome.

​ In order to test the function of P_AOX1, we construct "P_AOX1-α-factor-sfGFP-AOX1 terminator"(BBa_K3853050). If P_AOX1 is functional, we can test the fluorescence intensity of sfGFP in supernatant samples obtained from the culture of recombinant P.pastoris strain GS115.

​ Our results matched the general expected trend (Fig 1). After fermentation experiment in BMMY medium containing 0.5% methanol. The fluorescence intensity of the supernatant samples of recombinant P.pastoris strain GS115 containing the sfGFP gene gradually increased over time, while that of wild-type P.pastoris strain GS115 remained basically unchanged, which is in line with literature description^[1]. SDS-PAGE results (Fig 2) also verified this phenomenon, almost no protein band before 36 h could be seen. The corresponding protein band began to appear at 36 h, and the clarity and width of the protein band gradually increased over time, which means the AOX1 promoter can continuously induce the expression of the protein. At the same time, by measuring the growth curve of the strains (Fig 3), we observed that the OD₆₀₀ of the recombinant P.pastoris strain GS115 containing the sfGFP gene was slightly lower than the wild-type P.pastoris strain GS115 , the phenomenon of which may be attributed to the expression of sfGFP. The results showed that the expression of foreign genes would partly inhibit cell growth, but not in an intensive manner.

Fig. 1 Fluorescence intensity of supernatant samples obtained at different time points from the culture of wild-type P.pastoris strain GS115 and corresponding recombinant P.pastoris strain GS115 containing sfGFP gene.

Fig. 2 SDS-PAGE gel analysis of supernatant samples of the recombinant P.pastoris strain GS115 containing the sfGFP gene during the fermentation

 

Fig. 3 OD₆₀₀ absorbance obtained at different time points from the culture of wild-type P.pastoris strain GS115 and recombinant P.pastoris strain GS115 that contains sfGFP gene.

Reference

[1] Xuan, Y. et al. An upstream activation sequence controls the expression of AOX1 gene in Pichia pastoris. FEMS yeast research 9, 1271-1282, doi:10.1111/j.1567-1364.2009.00571.x (2009).