Part:BBa_K3239009:Experience

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Applications of BBa_K3239009

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

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

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

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

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

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

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

Figure 6. 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 AOX1 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.

• 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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