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Part:BBa_K3239010:Experience

Designed by: Xingyu Zhou   Group: iGEM19_ShanghaiFLS_China   (2019-10-16)
Revision as of 07:00, 18 October 2019 by Nicole Guo (Talk | contribs) (→‎Applications of BBa_K3239010)


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Applications of BBa_K3239010

This construct is designed to moderately up-regulate the AOX1 promoter (PAOX1) activity in P. pastoris GS115. It is to be integrated into the PAOX1-GFP strain. Upon methanol induction, PPRM1 expression of the homogeneous PRM1 gene is activated, and further amplified via the self-upregulation of Prm1. Prm1 then activates PMIT1, upregulating the expression of the homogeneous MIT1 gene and the heterogeneous PRM1 gene. This overall leads to a strong activation of PAOX1 expression of the AOX1 gene.

Unlike in wild type P. pastoris GS115 where MIT1 gene expression is then only upregulated by Prm1, in the constructed pAPM1-PAOX1-GFP strain, the heterogeneous MIT1 gene is also methanol induced as it is expressed by PPRM1. This corresponds to a much stronger activation PAOX1 compared to wild type P. pastoris GS115 upon initial methanol induction. In later stages, PAOX1 activation might be weaker due to an accumulation of Mit1 that suppresses PPRM1. After Mit1 is fully degraded, however, PPRM1 inhibition shall be removed and PAOX1 shall be again strongly induced. Therefore the regulation of PAOX1 activity should be more like a cyclical process.

We included self-inhibition of Mit1 in the construct as earlier research has demonstrated that if Mit1 is strongly expressed and self-upregulated by PAOX1 (which is activated by Mit1 and Prm1), the yeast will not survive. This suggests that Mit1 is slightly cytotoxic and its expression should be moderated at an appropriate level.

Given that in constructed strains, PAOX1 not only expresses the yEGFP3 reporter gene, but also the homogeneous AOX1 gene, the pAPM1-PAOX1-GFP strain should not only have a higher GFP yield compared to the PAOX1-GFP strain (see part BBa_K3239007) but also exhibit higher growth rates since it should be more capable of metabolizing methanol.

User Reviews

We labeled our strains containing this construct pAPM1-pAOX1-GFP and compared it to pAOX1-GFP, the positive control of our experiment.

Two identical pAPM1-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. pAPM1-pAOX1-GFP cell concentration curve during the 108h incubation period
Figure 2. pAPM1-pAOX1-GFP measured unit cell GFP production curve during the 108h incubation period
Figure 3. pAPM1-pAOX1-GFP aggregate unit cell GFP production curve during the 108h incubation period
Figure 4. pAPM1-pAOX1-GFP total GFP production curve during the 108h incubation period
Figure 5. pAPM1-pAOX1-GFP total methanol consumption during the 108h incubation period
Figure 6. pAPM1-pAOX1-GFP total GFP production per gram methanol during the 108h incubation period
Figure 6. pAPM1-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 raw 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 pAPM1-pAOX1-GFP strain, it is observed that of the two pAPM1-pAOX1-GFP constructs, pAPM1-pAOX1-GFP 1 exhibited very strong methanol induced GFP expression and its unit cell GFP expression surpassed that of pAOX1-GFP at virtually every time point during the 108h incubation period. In terms of total GFP production per gram methanol during the 108h incubation period, both pAPM1-pAOX1-GFP strains are at least at the same level as (pAPM1-pAOX1-GFP 2) or a much higher level than pAOX1-GFP (pAPM1-pAOX1-GFP 1). During the short-term methanol induction period, however, both strains have exhibited a unit cell GFP production at the same level as (pAPM1-pAOX1-GFP 2) or a much lower level than pAOX1-GFP (pAPM1-pAOX1-GFP 1). In an overview, however, pAPM1-pAOX1-GFP does have higher expression efficiency compared to the wildtype. 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 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, pPRM1 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 pPRM1 promoters AND the homogeneous pMIT1 promoter). This overall leads to a very strong activation of the pAOX1 promoter, which corresponds to a much higher expression level of GFP compared to AOX1-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 pAOX1 in pAPM1-pAOX1-GFP compared to pAOX1-GFP. Our data may be reflecting this process: during the 108h incubation period, Mit1 is much over-expressed and leads to a very strong activation of pAOX1. Yet later in the short-term methanol induction period, pAOX1 activation is very suppressed due to the compound effect of the suppression of pPRM1 by Mit1 and the degradation of Mit1. Nevertheless, after Mit1 is sufficiently degraded to derepress pPRM1, the over-expression should take place again. If the incubation is extended long enough, we may observe a much higher GFP expression level overall.


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