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        <h3 class="heading">Results and Analysis</h3>
 
 
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                 <p class="my-4">The main indicators of the GFP production efficiency in our experiment shall be the total GFP production per gram methanol during the 108 h 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.</p>
+
                 <p class="my-4">We cloned our plasmids <em>pGMP1</em> (carries <em>P<sub>MIT1</sub></em>-<em>PRM1</em> cassette) and <em>pAPM1</em> from the <em>pAPP1</em>(carries <em>P<sub>PRM1</sub></em>-<em>PRM1</em> cassette) and <em>pGMM1</em>(carries <em>P<sub>MIT1</sub></em>-<em>MIT1</em> cassette) plasmids used in the Wang et al., 2016 study, given to us as a gift from the ECUST school of Bioreactor Engineering. <em>(Thank you!)</em> The following diagram illustrates our construction process:</p>
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                <p class="my-4"><br />For the <em>pGMP1</em>-<em>P<sub>AOX1</sub></em>-GFP strain, it is evident from the data that during the 108 h incubation period, the expression efficiency of the construct started roughly at the same level as <em>pAOX1</em>-GFP but eventually surpassed the control. It is rather ambiguous whether the conversion rate from methanol to GFP in the <em>pGMP1</em>-<em>P<sub>AOX1</sub></em>-GFP strain after the 108 h incubation period is higher or lower than the control, yet during the short-term methanol induction period, the <em>pGMP1</em>-<em>P<sub>AOX1</sub></em>-GFP strain clearly has a much higher GFP expression level. This shows that <em>pGMP1</em>-<em>P<sub>AOX1</sub></em>-GFP does have a higher expression efficiency compared to the wildtype overall. </p>
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                <img src="https://static.igem.org/mediawiki/2019/5/59/T--ShanghaiFLS_China--Fig.456.png" alt="" class="img-fluid" />
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                 <p class="my-4"><br />When it comes to the <em>pAPM1</em>-<em>P<sub>AOX1</sub></em>-GFP strain, it is observed that of the two <em>pAPM1</em>-<em>P<sub>AOX1</sub></em>-GFP constructs, <em>pAPM1</em>-<em>P<sub>AOX1</sub></em>-GFP 1 exhibited very strong methanol induced GFP expression and its unit cell GFP expression surpassed that of <em>P<sub>AOX1</sub></em>-GFP at virtually every time point during the 108 h incubation period. In terms of total GFP production per gram methanol during the 108 h incubation period, both <em>pAPM1</em>-<em>P<sub>AOX1</sub></em>-GFP strains are at least at the same level as (<em>pAPM1</em>-<em>P<sub>AOX1</sub></em>-GFP 2) or a much higher level than PAOX1-GFP (<em>pAPM1</em>-<em>P<sub>AOX1</sub></em>-GFP 1). During the short-term methanol induction period, however, both strains have exhibited a unit cell GFP production at the same level as (<em>pAPM1</em>-<em>P<sub>AOX1</sub></em>-GFP2) or a much lower level than <em>P<sub>AOX1</sub></em>-GFP (<em>pAPM1</em>-<em>P<sub>AOX1</sub></em>-GFP1). In an overview, however, <em>pAPM1</em>-<em>P<sub>AOX1</sub></em>-GFP does have higher expression efficiency compared to the wildtype.</p>
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                <p class="p-citation"><strong>Plasmids construction scheme.</strong></p>
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                 <p class="my-4"><br />We fairly recently obtained our <em>pGMP1</em>-<em>pAPM1</em>-<em>P<sub>AOX1</sub></em>-GFP constructs and need to conduct more experiments to draw more conclusive results. In general, to yield more accurate data, 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.</p>
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                 <p class="my-4">We confirmed our constructions via a series of electrophoretic mobility shift assays:</p>
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         <h3 class="heading">Conclusions and Discussions</h3>  
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                <p class="p-citation"><strong>Electrophoretic mobility shift assays confirming the plasmid constructions.</strong></p>
 +
                <p class="my-4">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 <a href="https://2019.igem.org/Team:ShanghaiFLS_China/experiments"><u>experiments</u></a> page.</p>
 +
                <p class="my-4">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.</p>
 +
                <p class="my-4">When collecting data, we were aware that the activity of <em>P<sub>AOX1</sub></em> should not be reflected by the unit cell fluorescence intensity alone, as <em>P<sub>AOX1</sub></em> not only expresses GFP but also <em>AOX1</em>. While unit cell GFP production allows us to measure the expression of GFP, the expression of <em>AOX1</em> shall be represented by cell growth as yeasts in which more <em>AOX1</em> is expressed should also be more capable of metabolizing methanol, <em>i.e.</em> should be able to thrive more in methanol media. </p>
 +
                <p class="my-4">When collecting data, we were aware that the activity of <em>P<sub>AOX1</sub></em> should not be reflected by the unit cell fluorescence intensity alone, as <em>P<sub>AOX1</sub></em> not only expresses GFP but also <em>AOX1</em>. While unit cell GFP production allows us to measure the expression of GFP, the expression of <em>AOX1</em> shall be represented by cell growth as yeasts in which more <em>AOX1</em> is expressed should also be more capable of metabolizing methanol, <em>i.e.</em> should be able to thrive more in methanol media. </p>
 +
                <img src="https://static.igem.org/mediawiki/2019/a/a0/T--ShanghaiFLS_China--experiments_fig7.png" alt="" class="img-fluid" />
 +
                <p class="p-citation">(a) <strong>Measured unit cell GFP production curves during the 108h incubation period</strong>, 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) <strong>Measured cell concentration curves during the 108 h incubation period</strong>, where concentration Is expressed by OD<sub>600</sub>.</p>
 +
                <img src="https://static.igem.org/mediawiki/2019/5/5f/T--ShanghaiFLS_China--experiment_fig8.png" alt="" class="img-fluid" />
 +
                <p class="p-citation"><strong>Measured unit cell GFP production curves during the short-term methanol induction period</strong>, 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 OD<sub>600</sub>.</p>
 +
                 <p class="my-4"><br />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.</p>
 +
                <img src="https://static.igem.org/mediawiki/2019/d/d8/T--ShanghaiFLS_China--experiment_fig9.png" alt="" class="img-fluid" />
 +
                <p class="p-citation"><strong>Aggregate unit cell GFP production curves during the 108 h incubation period.</strong></p>
 +
                <p class="my-4">As mentioned before, <em>P<sub>AOX1</sub></em> 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 <em>P<sub>AOX1</sub></em> in each yeast strain.</p>
 +
                <img src="https://static.igem.org/mediawiki/2019/4/4f/T--ShanghaiFLS_China--experiment_fig10.png" alt="" class="img-fluid" />
 +
                <p class="p-citation"><strong>Total GFP production curves during the 108h incubation period.</strong></p>
 +
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         <h3 class="heading">References</h3>  
 
         <div class="row about-grids">  
 
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                 <p class="my-4">In general, our tested constructs do show a higher efficiency of methanol consumption compared to the wild type construct (<em>P<sub>AOX1</sub></em>-GFP). Of the four constructs tested, a 120% increase in total GFP production per gram methanol is exhibited <em>pAPM1</em>-<em>P<sub>AOX1</sub></em>-GFP in comparison to <em>P<sub>AOX1</sub></em>-GFP.</p>
+
                 <p class="my-4">Mateus, C., & Avery, S. V. (2000). Destabilized green fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry. <em>Yeast</em>, 16(14), 1313–1323. https://doi.org/10.1002/1097-0061(200010)16:14<1313::AID-YEA626>3.0.CO;2-O<br />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. <em>Journal of Biological Chemistry</em>, 291(12), 6245–6261. https://doi.org/10.1074/jbc.m115.692053</p>
−
                <p class="my-4">For <em>pGMP1</em>-<em>P<sub>AOX1</sub></em>-GFP, the reason for the protracted upregulation of <em>P<sub>AOX1</sub></em> may lie in the modified regulatory pathway itself. During the initial stages of methanol induction, <em>P<sub>PRM1</sub></em> is the major promoter being upregulated. Given that the homogeneous <em>P<sub>PRM1</sub></em>-<em>PRM1</em> cassette is unaltered and the introduced PRM1 is not expressed by <em>P<sub>PRM1</sub></em>, it is plausible that during these initial stages of methanol induction, <em>P<sub>AOX1</sub></em> is activated to approximately the same level in both <em>pGMP1</em>-<em>P<sub>AOX1</sub></em>-GFP and <em>P<sub>AOX1</sub></em>-GFP. In the later stages of methanol media incubation, however, <em>P<sub>MIT1</sub></em> begins to be activated by Prm1, which means that while the homogeneous Mit1 is upregulated to about the same level as in <em>P<sub>AOX1</sub></em>-GFP, the heterogeneous Prm1 is also upregulated. Moreover, whereas the homogenous expression of Prm1 is later suppressed by Mit1, the heterogeneous expression of Prm1 remains to be self-upregulated since <em>P<sub>MIT1</sub></em> is not inhibited like <em>P<sub>PRM1</sub></em>. In the end, the expression of both the Prm1 and Mit1 is much upregulated in <em>pGMP1</em>-<em>P<sub>AOX1</sub></em>-GFP compared to that of PAOX1-GFP.</p>
+
−
                <p class="my-4">For <em>pAPM1</em>-<em>P<sub>AOX1</sub></em>-GFP, on the other hand, the ostensibly self-contradicting results from the 108h incubation period and the short-term methanol induction period can be explained by the potentially cyclical nature of the modified regulation. In the initial stages of methanol induction, <em>P<sub>PRM1</sub></em> is activated, leading to an upregulation of both the homogeneous Prm1 and the heterogeneous Mit1, which is further amplified by the upregulation via Prm1 (Prm1 activates BOTH <em>P<sub>PRM1</sub></em> promoters <strong><em>AND</em></strong> the homogeneous <em>P<sub>MIT1</sub></em> promoter). This overall leads to a very strong activation of the <em>P<sub>AOX1</sub></em> promoter, which corresponds to a much higher expression level of GFP compared to <em>P<sub>AOX1</sub></em>-GFP. But later on, due to the accumulation of the over-expressed Mit1, the expression of both the homogenous Prm1 and the heterogeneous Mit1 are strongly suppressed, and consequently the expression of the homogenous Mit1 is also lowered as there's no Prm1 to activate it. This results in a much more suppressed activation of <em>P<sub>AOX1</sub></em> in <em>pAPM1</em>-<em>P<sub>AOX1</sub></em>-GFP compared to <em>P<sub>AOX1</sub></em>-GFP. Our data does reflect this process: during the 108 h incubation period, Mit1 is much over-expressed and leads to a very strong activation of <em>P<sub>AOX1</sub></em>. Yet later in the short-term methanol induction period, <em>P<sub>AOX1</sub></em> activation is very suppressed due to the compound effect of the suppression of <em>P<sub>PRM1</sub></em> by Mit1 and the degradation of Mit1. Nevertheless, after Mit1 is sufficiently degraded to derepress <em><sub>PPRM1</sub></em>, the over-expression should take place again. If the incubation is extended long enough, we may observe a much higher GFP expression level overall. Taken together, these results suggest that a co-culture of <em>pGMP1</em>-<em>P<sub>AOX1</sub></em>-GFP and <em>pAPM1</em>-<em>P<sub>AOX1</sub></em>-GFP may yield the highest methanol conversion rate throughout the incubation process.</p>
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Latest revision as of 14:12, 21 October 2019

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