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

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===Applications of BBa_K3239009===
 
===Applications of BBa_K3239009===
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We labeled our strains containing this construct pGMP1-pAOX1-GFP and compared it to pAOX1-GFP, the positive control of our experiment.  
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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.
  
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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.
  
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'''Methods'''
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Unlike in the wild type ''P. pastoris GS115'' where PRM1 gene expression is then inhibited by Mit1, in the ''pGMP1''-''P<sub>AOX1''-GFP strain, the heterogeneous ''PRM1'' gene is constantly self-upregulated due to the upregulation of ''P<sub>MIT1'' by Prm1. This leads to an overall upregulation of the expression of ''P<sub>AOX1'' transcription factors and hence shall further up-regulate ''P<sub>AOX1'' activity compared to wildtype ''P. pastoris GS115''.
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*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.
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*0.5%Methanol was added to the media every 24 hours to maintain the methanol concentration.
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*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.
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*Three parallels were performed for each strain.
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*We used a Biotek Synergy 2 plate reader to measure the GFP mean fluorescence intensities and the OD600 absorbance of our samples.
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Given that in constructed strains, ''P<sub>AOX1'' not only expresses the ''yEGFP3'' reporter gene, but also the homogeneous ''AOX1'' gene, the ''pGMP1''-''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.
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*Methanol concentration measurements were performed with a Shenzhen Sieman M100 Biosensors Analyzer.  
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[[File:Yeast Concentration(108h)pGMP1-pAOX1-GFP.png|200px|thumb|Figure 1. pGMP1-pAOX1-GFP cell concentration curve during the 108h incubation period]]
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===User Reviews===
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[[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]]
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For detailed modeling, design, experiment, results, and demonstration, please check our wiki:
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[[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]]
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[[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]]
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[[File:Total methanol consumption(108h)pGMP1-pAOX1-GFP.png|200px|thumb|Figure 5. pGMP1-pAOX1-GFP total methanol consumption during the 108h incubation period]]
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[[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]]
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[[File:Measured unit cell GFP(quick induction)pAOX1-GFP.png|200px|thumb|Figure 6. pGMP1-pAOX1-GFP measured unit cell GFP production curve during the short-term methanol induction period]]
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https://2019.igem.org/Team:ShanghaiFLS_China/Model
  
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'''Data'''
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https://2019.igem.org/Team:ShanghaiFLS_China/Design
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*OD600 is used as an indicator of yeast concentration in the experiment.
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*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 AOX1 at the sampled time point.
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*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.
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*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.
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*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.
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https://2019.igem.org/Team:ShanghaiFLS_China/Experiments
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https://2019.igem.org/Team:ShanghaiFLS_China/Results
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https://2019.igem.org/Team:ShanghaiFLS_China/Demonstrate
  
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*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!
 
  
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===User Reviews===
 
 
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Latest revision as of 06:59, 18 October 2019


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

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 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 the wild type P. pastoris GS115 where PRM1 gene expression is then inhibited by Mit1, in the pGMP1-PAOX1-GFP strain, the heterogeneous PRM1 gene 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 further up-regulate PAOX1 activity compared to wildtype P. pastoris GS115.

Given that in constructed strains, PAOX1 not only expresses the yEGFP3 reporter gene, but also the homogeneous AOX1 gene, the pGMP1-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

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


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