Part:BBa_K2944003

pTDH3-GOx-tPGK1

This coding sequences encodes a constitutive yeast promoter regulating expression of the glucose oxidase enzyme from Aspergillus niger. The gene sequence has been optimized for Saccharomyces cerevisiae. Glucose oxidase catalyzes the oxidation of glucose to D-glucono-1,5-lactone and hydrogen peroxide.

Characterization. Mass Spectrometry.

Activity of Glucose Oxidase was measured reacting the peroxide produce by the catalysis of glucose into gluconate with ammonium molybdate. The product of this reaction produces a yellow color that absorbs light at 405nm. The change in absorbance over time can be measured to determine if glucose oxidase is indeed present in the system.
Figure 1 – Absorbance of Cell Supernatant Over Time in the Presence of 100mM Ammonium Molybdate.

The sample was subjected to a solution of 10mM D-glucose and 100mM ammonium molybdate. The absorbance was measured periodically over a time frame of three hours.

Figure 2 Calibration Curve of the Absorbance at 405nm of Ammonium Molybdate

Table 1- Limit of Detection and Quantification of the Calibration Curve

<p>The change in absorbance indicates that glucose oxidase is present in the system and can be detected. Furthermore, the similarity in the rate of change in absorbance further suggests the presence of enzymatic activity. However, the level of peroxide detected is too low to be used to accurately quantified the concentration of peroxide in the system and the amount of glucose oxidase in the sample. This is likely due to the low cell concentration in the sample Which was approximately 1.74*107 cells/ml.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 717
Illegal BamHI site found at 1027
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1051
Illegal BsaI.rc site found at 2269