Team:UNSW Australia/Lab/Assays

Enzyme Assays

Biosynthesis of indole acetic acid (IAA) is the two-step enzymatic reaction we selected to use as proof of concept of our Assemblase scaffold. We used assays to determine the presence of the reactants and products in the Indole Acetic Acid (IAA) production pathway. The 3 reactants tested were tryptophan, Indole Acetamide (IAM), and IAA. We employed the Salkowski assay previously described by the 2011 Imperial College London iGEM team as a simple, fast, and inexpensive method of determining the amount of the final product IAA produced over a given time period. We employed HPLC to gather more detailed data about the relative abundance of each of the reactants and products over a given time period.

Aim

Our aim was to be able to determine the relative abundance of each of the reactants and final product in the IAA production pathway. In doing so, we hoped to evaluate the effectiveness of our scaffold by comparing the quantity of IAA produced by the scaffolded enzymes with the quantity of IAA produced by the non-scaffolded enzymes over a certain time period. Over the same time period, we expected a greater quantity of IAA to be detected when employing the scaffolded enzymes.

Salkowski Assay

The 2011 Imperial College London iGEM team describe a method for conducting a Salkowski assay on IAA. We have expanded upon their protocol.

Preparation of Salkowski Reagent (in fume hood):

  1. Measure 40mL of MQ H2O into a 100mL shot bottle with stirrer bar.
  2. Using a pipette, slowly transfer 60mL of H2SO4 (95%) into a 100mL measuring cylinder, ensuring the solution does not heat up excessively.
  3. Weigh 0.45g of anhydrous FeCl3 and add to acid solution with stirring until fully dissolved.
  4. Label and place in a dark corrosives-safe cabinet.

Preparation of 100mL 3mM stock IAA:

  1. Weigh 52.6mg IAA and dissolve in 5mL absolute ethanol.
  2. Add 5mL of the IAA ethanol solution to 95mL H2O.
  3. Dilute 1 in 10 for use in the reaction.

Reaction:

  • Add Salkowski Reagent to sample at a ratio of 2:1
  • Store in a dark corrosives-safe cupboard for 30 minutes.
  • Measure at OD530nm.

Salkowski Assay Results

We generated a standard curve using the assay, with an R2 of 0.99. Thus we were confident in our ability to detect IAA. The standard curve from the Salkowski assay is displayed in figure 1. The 300ul value was removed to generate the standard curve due to the assay being insensitive at concentrations above this level.

Figure 1: Salkowski assay standard curve. Values were run for 0ul, 50ul, 100ul, 150ul, and 250ul increments. (R2 = 0.99)

Figure 2: : Standard curve attained for ‘IAA concentration produced by vectors expressing IaaH and IaaM.’ (R2 = 0.99)

Figure 3: :A Cell based Salkowski Assay was performed on the supernatants of cells expressing IaaH and IaaM, incubated in the presence of tryptophan for varying time periods. Control denotes the vector contains no IaaH and IaaM producing gene. The concentration was produced by the ‘Figure 2’ standard curve, with upper limit of 250 uM, therefore, the insert value at 4hrs is inaccurate.

Figure 4: Cell based Salkowski assay performed on cell pellets following different durations of incubation with tryptophan.

Figure 5: IAM was incubated at different uM concentrations (Key) with the enzyme, IaaH (3.04ng/uL). All of the shown readings exceed the scale of the standard curve

Further Salkowski assays examining how readily the enzyme IaaH converted IAM to IAA showed us that the Salkowski reagent is not able to differentiate between the two reagents. Assays on cells expressing IAA pathway enzymes were also inconclusive. From this, we learnt that a more precise assay was required and so we opted to perform HPLC.

High Performance Liquid Chromatography (HPLC)

We present a method to prepare supernatant from lysed bacteria expressing the IaaM and IaaH enzymes and perform HPLC with these samples. This allowed for the measurement of tryptophan, indole acetamide, indole acetic acid over time.

  1. Acidify supernatant with 5M HCl to pH 2.5.
  2. Extract supernatant with an equal volume of ethyl acetate.
  3. Evaporate off the solvent by drying it through a vacuum (or leave it to evaporate).
  4. Dissolve in methanol & acetic acid --> pH 4.5
    1. 1mL of a solution of 25% methanol, 1% acetic acid, rest H2O.
    2. 250uL methanol, 10uL acetic acid, 740uL H2O
  5. Run system with solution:
    1. 72% of 1% acetic acid & 28% of 100% methanol.
    2. 0.8mL/minute with injection of 30.

HPLC Results

We were able to generate good standard curves for detection of each intermediary. The curves were generated from the chromatogram data returned by HPLC. Each curve displays the detection of each intermediary at concentrations of 5uM, 10uM, 25uM, 50uM, and 100 uM. Figure 2 displays the curve for Tryptophan, figure 3 displays the curve for IAM, and figure 4 displays the curve for IAA.

Figure 6: Standard curve for detection of Tryptophan. The peak area refers to the area under the chromatogram peak.

Figure 7: Standard curve for detection of indole acetamide. The peak area refers to the area under the chromatogram peak.

Figure 8: Standard curve for detection of Indole Acetic Acid. The peak area refers to the area under the chromatogram peak.

The following chromatograms display the ability of the method to detect the presence of each intermediary when mixtures are present. We report good mixture detection at 100uM and 10uM concentrations of each mixture (Figures 5 and 6), with less resolution when samples were present in 5uM concentrations (Figure 7).

Figure 9: Detection of the three intermediaries when present at 100uM. The peak at 20.994 corresponds to IAA, the peak at 19.203 corresponds to IAM, the peak at 15.200 corresponds to Tryptophan.

Figure 10: Detection of the three intermediaries when present at 10uM. The peak at 20.977 corresponds to IAA, the peak at 19.196 corresponds to IAM, the peak at 15.221 corresponds to Tryptophan.

Figure 11: Detection of the three intermediaries when present at 5uM. The peak at 20.986 corresponds to IAA, the peak at 19.186 corresponds to IAM, the peak at 15.210 corresponds to Tryptophan.

Discussion

The Salkowski assay is a suitable and relatively inexpensive tool to determine the concentration of IAA when it is pure in solution, giving a R2 of 0.994 for the initial IAA concentration determination. Problems arise when the assay is used to quantify IAA in the presence of IAM, as due to high absorbance observed when the Salkowski reagent is incubated with IAM; it is likely it the reagent also emits light at 530nm upon reacting with IAM. The Salkowski assay also suggests that within the cell based assays, most of the produced IAA remains in the pellet, thus the supernatant analysis may be redundant. These issues limit the Salkowski assay as a basic quantifier of IAA, thus it should only be used as a preliminary step to verify hypotheses about the production of IAA before more costly, effective and accurate methods such as HPLC are employed. HPLC analysis produced a far more accurate standard curves for IAA (R2 = 0.999), and very accurate curves for IAM (R2 = 1) and tryptophan (R2 = 1). HPLC allowed analysis of all three intermediates with one analysis, and has the potential to measure a changing concentration of these intermediates. Given the high quality and accuracy of standard results produced in the initial HPLC standards, more accurate analysis would provide little benefit as an assay for IAA.

Future direction

Further analysis of the changing concentration of IAA in IaaH/IaaM expressing cells incubated with tryptophan by HPLC could verify the potential of the enzymes to produce IAA from tryptophan. HPLC could then be used again, on scaffolded IaaH/IaaM incubated in the same conditions to determine any effect scaffolding these enzymes has on changing the rate of overall reaction in the IAA synthesis pathway.