Difference between revisions of "Part:BBa K3239010:Experience"

Latest revision as of 07:01, 18 October 2019

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Applications of BBa_K3239010

This construct is designed to moderately up-regulate the AOX1 promoter (P_AOX1) activity in P. pastoris GS115. It is to be integrated into the P_AOX1-GFP strain. Upon methanol induction, P_PRM1 expression of the homogeneous PRM1 gene is activated, and further amplified via the self-upregulation of Prm1. Prm1 then activates P_MIT1, upregulating the expression of the homogeneous MIT1 gene and the heterogeneous PRM1 gene. This overall leads to a strong activation of P_AOX1 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-P_AOX1-GFP strain, the heterogeneous MIT1 gene is also methanol induced as it is expressed by P_PRM1. This corresponds to a much stronger activation P_AOX1 compared to wild type P. pastoris GS115 upon initial methanol induction. In later stages, P_AOX1 activation might be weaker due to an accumulation of Mit1 that suppresses P_PRM1. After Mit1 is fully degraded, however, P_PRM1 inhibition shall be removed and P_AOX1 shall be again strongly induced. Therefore the regulation of P_AOX1 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 P_AOX1 (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, P_AOX1 not only expresses the yEGFP3 reporter gene, but also the homogeneous AOX1 gene, the pAPM1-P_AOX1-GFP strain should not only have a higher GFP yield compared to the P_AOX1-GFP strain (see part BBa_K3239007) but also exhibit higher growth rates since it should be more capable of metabolizing methanol.

User Reviews

For detailed modeling, design, experiment, results, and demonstration, please check our wiki:

https://2019.igem.org/Team:ShanghaiFLS_China/Model

https://2019.igem.org/Team:ShanghaiFLS_China/Design

https://2019.igem.org/Team:ShanghaiFLS_China/Experiments

https://2019.igem.org/Team:ShanghaiFLS_China/Results

https://2019.igem.org/Team:ShanghaiFLS_China/Demonstrate

UNIQ5605cef6b6c8f46d-partinfo-00000000-QINU UNIQ5605cef6b6c8f46d-partinfo-00000001-QINU

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 ===Applications of BBa_K3239010===
-We labeled our strains containing this construct pGMP1-pAOX1-GFP and compared it to pAOX1-GFP, the positive control of our experiment.
+This construct is designed to moderately up-regulate the ''AOX1'' promoter (''P<sub>AOX1'') activity in ''P. pastoris GS115''. It is to be integrated into the ''P<sub>AOX1''-GFP strain. Upon methanol induction, ''P<sub>PRM1'' expression of the homogeneous ''PRM1'' gene is activated, and further amplified via the self-upregulation of Prm1. Prm1 then activates ''P<sub>MIT1'', upregulating the expression of the homogeneous ''MIT1'' gene and the heterogeneous ''PRM1'' gene. This overall leads to a strong activation of ''P<sub>AOX1'' expression of the ''AOX1'' gene.
-Two identical pGMP1-pAOX1-GFP strains were used in our experiment, labeled 1 and 2 respectively.
+Unlike in wild type ''P. pastoris GS115'' where ''MIT1'' gene expression is then only upregulated by Prm1, in the constructed ''pAPM1''-''P<sub>AOX1''-GFP strain, the heterogeneous ''MIT1'' gene is also methanol induced as it is expressed by ''P<sub>PRM1''. This corresponds to a much stronger activation ''P<sub>AOX1'' compared to wild type ''P. pastoris GS115'' upon initial methanol induction. In later stages, ''P<sub>AOX1'' activation might be weaker due to an accumulation of Mit1 that suppresses ''P<sub>PRM1''. After Mit1 is fully degraded, however, ''P<sub>PRM1'' inhibition shall be removed and ''P<sub>AOX1'' shall be again strongly induced. Therefore the regulation of ''P<sub>AOX1'' 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 ''P<sub>AOX1'' (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.
-'''Methods'''
+Given that in constructed strains, ''P<sub>AOX1'' not only expresses the ''yEGFP3'' reporter gene, but also the homogeneous ''AOX1'' gene, the ''pAPM1''-''P<sub>AOX1''-GFP strain should not only have a higher GFP yield compared to the ''P<sub>AOX1''-GFP strain (see part BBa_K3239007) but also exhibit higher growth rates since it should be more capable of metabolizing methanol.
-*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.
+===User Reviews===
-*Methanol concentration measurements were performed with a Shenzhen Sieman M100 Biosensors Analyzer.
+For detailed modeling, design, experiment, results, and demonstration, please check our wiki:
-[[File:Yeast Concentration(108h)pGMP1-pAOX1-GFP.png|200px|thumb|Figure 1. pGMP1-pAOX1-GFP cell concentration curve during the 108h incubation period]]
+https://2019.igem.org/Team:ShanghaiFLS_China/Model
-[[File:Measured unit cell GFP(108h)pGMP1-pAOX1-GFP.png|200px|thumb|Figure 2. pGMP1-pAOX1-GFP measured unit cell GFP production curve during the 108h incubation period]]
-[[File:Aggregate unit cell GFP(108h)pGMP1-pAOX1-GFP.png|200px|thumb|Figure 3. pGMP1-pAOX1-GFP aggregate unit cell GFP production curve during the 108h incubation period]]
-[[File:Total GFP production(108h)pGMP1-pAOX1-GFP.png|200px|thumb|Figure 4. pGMP1-pAOX1-GFP total GFP production curve during the 108h incubation period]]
-[[File:Total methanol consumption(108h)pGMP1-pAOX1-GFP.png|200px|thumb|Figure 5. pGMP1-pAOX1-GFP total methanol consumption during the 108h incubation period]]
-[[File:Total GFP production per gram methanol(108h)pGMP1-pAOX1-GFP.png|200px|thumb|Figure 6. pGMP1-pAOX1-GFP total GFP production per gram methanol during the 108h incubation period]]
-[[File:Measured unit cell GFP(quick induction)pGMP1-pAOX1-GFP.png|200px|thumb|Figure 6. pGMP1-pAOX1-GFP measured unit cell GFP production curve during the short-term methanol induction period]]
+https://2019.igem.org/Team:ShanghaiFLS_China/Design
-'''Data'''
+https://2019.igem.org/Team:ShanghaiFLS_China/Experiments
-*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 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.
+https://2019.igem.org/Team:ShanghaiFLS_China/Results
-*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!
+https://2019.igem.org/Team:ShanghaiFLS_China/Demonstrate
-'''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 our construct does have a higher expression efficiency compared to the wildtype construct overall.
-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 heterogeneous Prm1 and consequently the homogenous Mit1 is much upregulated in pPRM1-pAOX1-GFP compared to the pAOX1-GFP.
-===User Reviews===
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