Part:BBa_K3239007

pAOX1-GFP

yEFGP3 reporter gene expressed by pAOX1

Usage and Biology

This construct is the control used in our experiment. After homologous recombination into wild type P. pastoris, the EGFP expressed shall serve as an indicator of AOX1 expression levels. Note that the original pAOX1-AOX1 copy remains intact after the recombination.

Experience

we labeled our strains containing this construct AOX1-GFP and used it as the positive control of our data.

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.
• 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. 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 0.5% methanol, 50µg/mL histidine and 0.4µg/mL biotin) for 12 hours and sampled every 2 hours. The starting concentration was 3 OD600.
• Three parallels were performed for each strain.

Figure 1. AOX1-GFP cell concentration curve during the 108h incubation period

Figure 2. AOX1-GFP measured unit cell GFP curve during the 108h incubation period

• We used a Biotek Synergy 2 plate reader to measure the GFP geometric-mean intensities and the OD600 absorbance of our samples.
• Methanol concentration measurements were performed with a Shenzhen Sieman M100 Biosensors Analyzer.

• OD600 is used as an indicator of yeast concentration in the sample.
• Measured unit cell GFP is the ratio of the geometric mean of the GFP 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 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.

Source

Both the promoter and the reporter are registered. pAOX1: BBa_K3239003 yEGFP3: BBa_K3239006

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 935
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]