Team:Berkeley/Methods

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Purpose:

The Berkeley iGEM team 2013 has determined the enzyme kinetics of FMO and GLU (data for: FMO and GLU) based on the Michaelis-Menten kinetics model. Our purpose of determining kinetics was to give a quantitative data of how fast these enzymes can turnover their respective substrates, which is very useful when considering scaling up the process and for future iGEM teams when they use our enzymes.

Materials:

• Purified Enzyme: FMO or GLU
• Substrate:
  • FMO: Indole, NADPH (co-factor)
  • GLU: Indican
• TECAN Plate Reader and TECAN Plate
• Solvents: DMSO, Water

Methods:

1. Determining the Concentration of Purified Enzyme

Note: We followed the protocol from Bio-Rad’s Bradford Assay to determine the concentration of purified FMO and GLU.

We determined the concentration of our purified enzyme to be 4.2 mg/L for FMO and 3.5 mg/L for GLU.

2. Determining the Michaelis Menten kinetics

Note: We assumed that the oxidation of indoxyl to indigo is much faster than that of the turnover rate of indole to indoxyl by FMO.