Difference between revisions of "Part:BBa K2944003"

Revision as of 22:06, 21 October 2019

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 by Mass Spectrometry.

In HPLC-MS, samples are purified using HPLC and are then analyzed using mass spectroscopy to determine the compound. This system can not only identify samples with high accuracy, but also quantitate them based on the area of the peak producing the signal of interest.

Preparation of Standard Curve

https://2019.igem.org/wiki/images/8/8f/T--Concordia-Montreal--GoxCharacterization.pdf

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

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.

Experimental

https://2019.igem.org/wiki/images/6/6b/T--COncordia-Montreal--massspect.pdf Table 1- Experimental Conditions

Sample prep

Standards

Authentic gluconolactone and gluconate standards were purchased from Sigma-Aldrich

Samples

Samples diluted 1:5 in cold acetonitrile and centrifuged at 21,000 RCF for 2 minutes

5 μL was injected into HPLC

HPLC Conditions

Machine:

1290 Infinity II LC system (Agilent Technologies)

Column:

Zorbax Eclipse Plus C18 column (50 x 2.1 mm, 1.8 uM; Agilent Technologies)

Column Temperature:

30°C

Solvents:

A: 0.1% formic acid in water

B: 0.1% formic acid in acetonitrile

Gradient:

2%B for 1 min, 85%B for 2 min, followed by re-equilibration for 2 min

MS Conditions

Machine:

Agilent 6545 quadrupole time-of-flight MS (QTOF-MS; Agilent Technologies)

Gas Settings:

Sheath gas flow rate: 10 L / min

Sheath gas temperature: 350 C

Drying gas flow rate: 12 L / min

Drying gas temperature: 325 C

Nebulizing gas: 55 psig
Ionization

Negative mode

Sample Analysis

Programs:

Agilent MassHunter Qualitative Analysis software

Agilent MassHunter Quantitative Analysis software

Compound confirmation

Exact mass of gluconolactone, [M-H]- : 177.0399

Exact mass of gluconate, [M-H]- : 195.0505

Samples were confirmed by comparison of retention time and exact mass to authentic standard

Exact mass was accurate to < 10 ppm

In HPLC-MS, samples are purified using HPLC and are then analyzed using mass spectroscopy to determine the compound. This system can not only identify samples with high accuracy, but also quantitate them based on the area of the peak producing the signal of interest.

Table 1Calibration Data


Figure 1-Figure 1- Standard Curves of Gluconolactone and Gluconate

Table 3- Sample Signals and Determined Concentration


Both samples show the production of gluconate although these values are low, they are still able to be quantified within the range of the calibration curve. The most likely reason for low production is a population of cells in the sample.

Sequence and Features