Team:Berkeley/Project/FMO

Flavin-containing monooxygenase (FMO) is the enzyme responsible for indigo formation through the conversion of indole to indoxyl with NADPH as its co-factor. Under the presence of oxygen, indoxyl spontaneously dimerizes to indigo.

One of our project goals this year is to improve the characterization of our indigo producing part so that other iGEM teams in the future can have access to this reliable and well characterized part. In order to characterize this enzyme, we looked to verify whether we were actually producing indigo, checked for factors affecting bio-indigo synthesis, and calculated kinetic data for our enzyme.

An important aspect in characterizing our FMO enzyme is to look into factors affecting indigo synthesis in order to be able to titrate the amount of indigo being produced. To do this, we ran a series of experiments with varying levels of tryptophan, pH buffering, and differing salts.

After culturing E. coli cells in plain LB media with resistance for over 24 hours, we quantified indigo concentration to be approximately 2.06 mg/L. Once we increased the tryptophan concentration in the media, we got an appreciable increase in bio-indigo production. We also tried two different salts, KCl and NaCl due to previous research suggesting potassium and sodium ions have an effect on the enzyme tryptophanase (tnaA), one of the enzymes involved in converting tryptophan to indole(superscript). However, there was little to no difference in indigo production from the different salts. Additionally, after buffering the pH level with sodium phosphate at pH 7, we observed little to no difference in indigo production.

From this, we concluded that the main factor affecting indigo production was the level of tryptophan present in the media. This makes sense because this is the raw material for cells in order to produce indole, the substrate the FMO enzyme acts on. The maximum amount of indigo we were able to produce was approximately 224 mg/L in buffered conditions and 2.4 g/L tryptophan concentration in the media. The results of these titrations are summarized below.

Experiment Scheme/Limitations

Cells were cultured in 5ml glass tubes in the appropriate media at 30o C for over 24 hours. YE stands for yeast extract. Buffering was done by the addition of sodium phosphate. Refreshment refers to the replacement of media after approximately 13 hours of growth.

Indigo Quantification

All indigo quantifications were done through absorbance measurements at 620 nm with TECAN instrument. In order to quantify our indigo production using this method, we created a standard indigo calibration curve with a stock solution of pure indigo in dimethyl sulfoxide (DMSO). The results of the calibration curve are below with a R2 value of 0.994. The calibration curve used in the experiment is shown below.

Additionally, we wanted to measure how quickly FMO generated indigo from its substrate, indole. We used Michaelis-Menten kinetics to model the behavior of FMO with various concentrations of indole substrate to determine the Km and Vmax of the enzyme.

To generate kinetic data for FMO, we first needed to obtain purified enzyme. We used the T7 expression system and chemically competent BL21 cells to express a His6 tagged FMO and purify it using a nickel column. With this purified enzyme, we ran an assay with different concentrations of indole in a mixture containing 0.1 mM NADPH, 0.1 mM EDTA, and Bicine/KOH buffer (pH 8.5).



From this data, we generated a Michaelis-Menten kinetics graph, and found the Vmax and Km to be 1.166*10^(-5) mM/s and 0.8698 mM respectively. At the 95% confidence level, Vmax is in the range of [0.00000935 mM/s, 0.00001398 mM/s], while Km is in the range of [0.12 mM, 1.62 mM].

Experiment Scheme/Limitations

The kinetics assay was adopted from previous work with FMO on substrates such as trimethylamine (2 ref). To our knowledge, this is the first kinetic assay done with indole. All kinetic assays were carried out measuring absorbance at 620 nm with TECAN. A limitation in our kinetic assay is that we assumed that the oxidation step from indoxyl to indigo was nearly instantaneous, and subsequently measured only the indigo concentration. However, we believe that because of sufficient aeration and oxygen content during our assay, the error coming from this assumption would be very limited.

We were also interested in whether over-accumulation of indigo would be toxic to E. coli cells. This was of particular interest to us because large scale production of bio-indigo if toxicity occurred. To test the toxicity of indigo, we ran a time course experiment in which we measured the number of bacteria over a 24 hour period. Cells with the FMO plasmid and mutant FMO plasmid were cultured in 5ml tubes with 0.74 g/L tryptophan at 30 C. The mutant FMO, which contains mutagenized catalytic sites of the FMO, served as a control for this experiment. From this experiment, we observed no noticeable toxicity with indigo.

Limitations of Growth Assay