Difference between revisions of "Part:BBa K3239009:Experience"

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Applications of BBa_K3239009

This construct is designed to moderately up-regulate the AOX1 promoter (pAOX1) activity in P. pastoris GS115. It is to be homologously recombined into the pAOX1-GFP strain. Upon methanol induction, pPRM1 expression of the homogeneous Prm1 is activated, and further amplified via the self-upregulation of Prm1. Prm1 then activates pMIT1, upregulating the expression of the homogeneous Mit1 and the heterogeneous Prm1.

Unlike wildtype P. pastoris GS115 where Prm1 expression is then inhibited by Mit1, in the constructed pGMP1-pAOX1-GFP strain, the heterogeneous Prm1 is constantly self-upregulated due to the upregulation of pMIT1 by Prm1. This leads to an overall upregulation of the expression of pAOX1 transcription factors and hence shall up-regulate pAOX1 activity compared to wildtype P. pastoris.

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.

User Reviews

We labeled our strains containing this construct pGMP1-pAOX1-GFP and compared it to pAOX1-GFP, the positive control of our experiment.

Two identical pGMP1-pAOX1-GFP strains were used in our experiment, labeled 1 and 2 respectively.

Methods

• The following data were acquired after 108h 50mL YNM (1.34% yeat nitrogenous base with 0.5% methanol, 50µg/mL histidine and 40µg/mL biotin) incubation, sampled every 12h. The starting concentration was 1 OD600.
• 0.5%Methanol was added to the media every 24 hours to maintain the methanol concentration.
• A short-term methanol induction experiment was performed after the 108h incubation to characterize the instant response to methanol induction after the yeast cells are accustomed to methanol media, hence indicating the overall induction activity of the construct. The yeast cells were collected, rinsed and stored at 4˚C overnight to allow GFP to fully degrade. The strains were then incubated in higher-concentration 50mL YNM media (1.34% yeat nitrogenous base with 0.75% methanol, 50µg/mL histidine and 40µg/mL biotin) for 12 hours and sampled every 2 hours. The starting concentration was 3 OD600.
• Three parallels were performed for each strain.

• We used a Biotek Synergy 2 plate reader to measure the GFP mean fluorescence intensities and the OD600 absorbance of our samples.
• Methanol concentration measurements were performed with a Shenzhen Sieman M100 Biosensors Analyzer.

Figure 1. pGMP1-pAOX1-GFP cell concentration curve during the 108h incubation period

Figure 2. pGMP1-pAOX1-GFP measured unit cell GFP production curve during the 108h incubation period

Figure 3. pGMP1-pAOX1-GFP aggregate unit cell GFP production curve during the 108h incubation period

Figure 4. pGMP1-pAOX1-GFP total GFP production curve during the 108h incubation period

Figure 5. pGMP1-pAOX1-GFP total methanol consumption during the 108h incubation period

Figure 6. pGMP1-pAOX1-GFP total GFP production per gram methanol during the 108h incubation period

Figure 6. pGMP1-pAOX1-GFP measured unit cell GFP production curve during the short-term methanol induction period

Data

• OD600 is used as an indicator of yeast concentration in the experiment.
• Measured unit cell GFP production is the ratio of the GFP mean fluorescence intensities to the 0D600 absorbance measured by the plate reader. It shall serve as an indicator of the expression level of GFP at the sampled time point.
• Aggregate unit cell GFP production is the calculated total unit cell GFP fluorescence intensity. It accounts for the GFP that has degraded over the incubation period to more accurately reflect the aggregate production rate.
• Total GFP production is the product of the yeast concentration and the aggregate unit cell GFP. It reflects the total GFP production of the reaction system of the strain.
• Total GFP production per gram methanol is the ratio of the total GFP production to the total methanol consumption. It reflects the overall conversion rate of methanol to the product.

• For teams wishing to compare their data to ours, we've provided our data along with our plate reader calibration data in our wiki. Feel free to give it a check!

Conclusions and Discussions

The main indicators of the GFP production efficiency in our experiment shall be the total GFP production per gram methanol during the 108h incubation period and the measured unit cell GFP production during the short-term methanol induction period, as the former demonstrates the overall conversion rate from the substrate to the desired product over a fairly extended period, while the latter demonstrates the instantaneous expression efficiency of the construct after stabilization in the methanol media.

For the pGMP1-pAOX1-GFP strain, it is evident from the data that during the 108h incubation period, the expression efficiency of the construct started roughly at the same level as pAOX1-GFP but eventually surpassed the control. It is rather ambiguous whether the conversion rate from methanol to GFP in the pGMP1-pAOX1-GFP strain after the 108h incubation period is higher or lower than the control, yet during the short-term methanol induction period, the pGMP1-pAOX1-GFP strain clearly has a much higher GFP expression level. This shows that pGMP1-pAOX1-GFP does have a higher expression efficiency compared to the wildtype overall. To yield more accurate data, however, incubation over a further extended period in a more stable system (e.g.week-long chemostat incubation) and the substitution of GFP with a more stable reporter might be necessary.

The reason for the protracted upregulation of pAOX1 may lie in the modified regulatory pathway itself. During the initial stages of methanol induction, pPRM1 is the major promoter being upregulated. Given that the homogeneous pPRM1-PRM1 cassette is unaltered and the introduced PRM1 is not expressed by pPRM1, it is plausible that during these initial stages of methanol induction, pAOX1 is activated to approximately the same level in both pGMP1-pAOX1-GFP and pAOX1-GFP. In the later stages of methanol media incubation, however, pMIT1 begins to be activated by Prm1, which means that while the homogeneous Mit1 is upregulated to about the same level as in pAOX1-GFP, the heterogeneous Prm1 is also upregulated. Moreover, whereas the homogenous expression of Prm1 is later suppressed by Mit, the heterogeneous expression of Prm1 remains to be self-upregulated since pMIT1 is not inhibited like pPRM1. In the end, the expression of both the Prm1 and Mit1 is much upregulated in pPRM1-pAOX1-GFP compared to that of pAOX1-GFP.

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