Team:ShanghaiFLS China/Results
We cloned our plasmids pGMP1 (carries PMIT1-PRM1 cassette) and pAPM1 from the pAPP1(carries PPRM1-PRM1 cassette) and pGMM1(carries PMIT1-MIT1 cassette) plasmids used in the Wang et al., 2016 study, given to us as a gift from the ECUST school of Bioreactor Engineering. (Thank you!) The following diagram illustrates our construction process:
Plasmids construction scheme.
We confirmed our constructions via a series of electrophoretic mobility shift assays:
Electrophoretic mobility shift assays confirming the plasmid constructions.
We conducted multiple rounds of exhaustive experiments as we wanted to try our best to simulate industrial fermenter productions with our limited equipment. In our last two rounds we conducted incubations over a timescale of 108 hours and sampled at regular intervals for analysis. We also conducted a follow-up experiment for further understanding of our constructs. The protocols of this experiment and reasons behind these decisions can be found on our experiments page.
For each reaction system, 2~3 parallels were performed. We did the 108h incubation twice and the results from the two rounds corroborate each other. The following data stems from the second round of 108 h incubation as we collected more data in this round.
When collecting data, we were aware that the activity of PAOX1 should not be reflected by the unit cell fluorescence intensity alone, as PAOX1 not only expresses GFP but also AOX1. While unit cell GFP production allows us to measure the expression of GFP, the expression of AOX1 shall be represented by cell growth as yeasts in which more AOX1 is expressed should also be more capable of metabolizing methanol, i.e. should be able to thrive more in methanol media.
When collecting data, we were aware that the activity of PAOX1 should not be reflected by the unit cell fluorescence intensity alone, as PAOX1 not only expresses GFP but also AOX1. While unit cell GFP production allows us to measure the expression of GFP, the expression of AOX1 shall be represented by cell growth as yeasts in which more AOX1 is expressed should also be more capable of metabolizing methanol, i.e. should be able to thrive more in methanol media.
(a) Measured unit cell GFP production curves during the 108h incubation period, where the measured unit cell GFP production is the ratio of the mean of the measured GFP fluorescence intensities to the measured cell concentration expressed by OD600. (b) Measured cell concentration curves during the 108 h incubation period, where concentration Is expressed by OD600.
Measured unit cell GFP production curves during the short-term methanol induction period, where the measured unit cell GFP production is the ratio of the mean of the measured GFP fluorescence intensities to the measured cell concentration expressed by OD600.
During the 108 h incubation period, while the measured cell concentrations accounts for all the cell that has ever existed in the flask (the amount sampled is considered negligible), the measured unit cell GFP production do not account for all the GFP that has been produced due to the fact that GFP degrades over time, and its half-life might be very different from the produce in industrial applications. We therefore ruled out the effect of GFP degradation by accounting for its half-life (~7 h as reported in Mateus & Avery, 2000), which yields the aggregate unit cell GFP production.
Aggregate unit cell GFP production curves during the 108 h incubation period.
As mentioned before, PAOX1 activity should not only be represented by GFP production but also by cell growth, we calculated the total GFP production of each reaction system by multiplying aggregate unit cell GFP production and cell concentrations and the total volume of the media. These data shall most comprehensively reflect the upregulation of PAOX1 in each yeast strain.
Total GFP production curves during the 108h incubation period.
References
Mateus, C., & Avery, S. V. (2000). Destabilized green fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry. Yeast, 16(14), 1313–1323. https://doi.org/10.1002/1097-0061(200010)16:14<1313::AID-YEA626>3.0.CO;2-O
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