In order to determine the successful activity of our chosen enzymes, we used a number of assays to measure levels of reactants and products in the indole-3-acetic acid (IAA) production pathway. Assays were designed to measure the presence of tryptophan (initial reactant), indole-3-acetamide (IAM, the intermediate reactant) and IAA (product). As a simple, fast, and inexpensive test we used a Salkowski assay, previous described by iGEM Imperial College London 2011, which measured levels of IAA production over time. An HPLC assay was additionally used to gather more detailed data about the relative abundance of each of the reactants and products over a given time period.
IAA biosynthesis was selected as our candidate pathway for investigating the performance of our Assemblase scaffold. The IAA biosynthesis pathway involves the conversion of tryptophan to indole-3-acetamide by the enzyme IaaM, followed by conversion to IAA by the enzyme IaaH (Figure 1). In order to examine the performance of the IaaM and IaaH enzymes in our Assemblase scaffolding system compared to when they are free in solution, it was necessary to set up assays to quantify reaction turnover. To this aim, we established the Salkowski assay and HPLC in our lab. These assays allow us to determine the relative abundance of each of the reactants and final product in the IAA production pathway.
Figure 1: The indole-3-acetic acid (IAA) biosynthesis pathway.
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.
The Salkowski assay is a colourimetric assay that detects IAA in solution. This simple assay was therefore useful to compare IAA production by unscaffolded veruses Assemblase-scaffolded IaaM and IaaH enzymes. We first performed the Salkowski assay on standards prepared from commercially available IAA. The 2011 Imperial College London iGEM team described a method for conducting a Salkowski assay on IAA, we have expanded their method.
Preparation of Salkowski Reagent (in fume hood):
- Measure 40 mL of MQ H2O into a 100 mL shot bottle with stirrer bar.
- Using a pipette, slowly transfer 60mL of H2SO4 (95%) into a 100 mL measuring cylinder, ensuring the solution does not heat up excessively.
- Weigh 0.45 g of anhydrous FeCl3.
- Add to acid solution with stirring until fully dissolved.
- Label and place in a dark corrosives-safe cabinet.
Preparation of 100 mL 3 mM stock IAA:
- Weigh 52.6 mg IAA and dissolve in 5 mL absolute ethanol.
- Add 5 mL of the IAA ethanol solution to 95 mL H2O.
- Dilute 1 in 10 for use in the reaction.
Reaction (Non cell based analysis):
- Add Salkowski Reagent to sample at a ratio of 2:1
- Store in a dark corrosives-safe cupboard for 30 minutes.
- Measure at OD530nm.
Reaction (Cell based analysis):
- Centrifuge cell suspensions intended for analysis for 5 minutes on maximum speed.
- Analyse the supernatant as per ‘Non cell based analysis’ protocol.
- Remove all of the supernatant from the pellet
- Add 1mL of Salkowski reagent to the pellet, then 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 (Figures 2 and 3). The 300 µl value was removed to generate the standard curve due to the concentration being beyond the detection limit of this assay.
Figure 2: Salkowski assay standard curve. Values were run for 0 µl, 50 µl, 100 µl, 150 µl, and 250 µl increments. (R2 = 0.99)
Figure 3: Standard curve attained for ‘IAA concentration produced by cells expressing IaaM and IaaH.’ (R2 = 0.99)
Escherichia coli T7 Express cells producing IaaM and IaaH were grown in tryptophan-enriched Luria broth, and Salkowski assays were performed on cell pellets collected and the resulting supernatant (Figures 4 and 5). However, these were inconclusive due to contamination of the control sample. Salkowski assays to determine how readily the purified enzyme IaaH converted IAM to IAA showed us that the Salkowski reagent is not able to differentiate between the two molecules (Figure 6). From this, we learnt that a more precise assay was required and so we opted to perform HPLC.
Figure 4: A Cell based Salkowski Assay was performed on the supernatants of cells expressing IaaM and IaaH, incubated in the presence of tryptophan for varying time periods. Control denotes the vector contains no IaaM and IaaH producing gene. The concentration was produced by the ‘Figure 3’ standard curve, with upper limit of 250 µM, therefore, the insert value at 4 hrs is inaccurate.
Figure 5: Cell based Salkowski assay performed on cell pellets following different durations of incubation with tryptophan.
Figure 6: IAM was incubated at different µM concentrations (Key) with the enzyme, IaaH (3.04 ng/µL). All of the shown readings exceed the scale of the standard curve
High Performance Liquid Chromatography (HPLC)
High Performance Liquid Chromatography (HPLC) is a technique used to analyse, measure, separate and identify components of a mixture. The mixture passes through a column packed with an adsorbent material, which causes different flow rates for the different components of the mixture. We present a method to prepare the supernatant from lysed cells containing the enzymes. This allows for the measurement of the 3 key intermediaries using HPLC; tryptophan, indole acetamide, indole acetic acid. HPLC enables measurement of these intermediaries with greater sensitivity than the Salkowski assay and is able to distinguish between indole acetamide and indole acetic acid.
- Acidify supernatant with 5M HCl to pH 2.5.
- Extract supernatant with an equal volume of ethyl acetate.
- Evaporate off the solvent by drying it through a vacuum (or leave it to evaporate).
- Dissolve in methanol & acetic acid (pH 4.5)
- 1 mL of a solution of 25% methanol, 1% acetic acid, rest H2O.
- 250 µL methanol, 10 µL acetic acid, 740 µL H2O
- Run system with solution:
- 72% of 1% acetic acid & 28% of 100% methanol.
- 0.8 mL/minute with injection of 30 µL.
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 5 µM, 10 µM, 25 µM, 50 µM, and 100 µM (Figures 7, 8, and 9).
Figure 7: Standard curve for detection of tryptophan. The peak area refers to the area under the chromatogram peak.
Figure 8: Standard curve for detection of indole acetamide. The peak area refers to the area under the chromatogram peak.
Figure 9: Standard curve for detection of indole acetic acid. The peak area refers to the area under the chromatogram peak.
HPLC chromatograms were recovered, demonstrating the ability of the method to detect the presence of each intermediary in mixed solutions. We report good mixture detection at 100 µM and 10 µM concentrations of each mixture , with less resolution when samples were present in 5 µM concentrations (Figures 10, 11, and 12).
Figure 10: Detection of the three intermediaries when present at 100 µM. 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 11: Detection of the three intermediaries when present at 10 µM. 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 12: Detection of the three intermediaries when present at 5 µM. The peak at 20.986 corresponds to IAA, the peak at 19.186 corresponds to IAM, the peak at 15.210 corresponds to Tryptophan.
The Salkowski assay is a suitable and relatively inexpensive tool to determine the concentration of IAA when it is pure in solution. Our Salkowski assay standard curve for IAA concentration generated an R2 value of 0.994. Unfortunately our results using IaaM and IaaH expressing bacterial cell pellets and supernatant were inconclusive due to contamination of the control sample (Figures 4 and 5). Further Salkowski assays to determine how readily purified enzyme IaaH converted IAM to IAA showed us that the Salkowski reagent is not able to differentiate between the two molecules (Figure 6). From this, we learnt that a more precise assay was required and so we opted to perform HPLC.
HPLC analysis produced far more accurate standard curves for IAA (R2 = 0.999), and very accurate curves for IAM (R2 = 1.000) and tryptophan (R2 = 1.000). 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.
Further analysis of the changing concentration of IAA in IaaM/IaaH 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 IaaM/IaaH 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.