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Team:Exeter/enzyme-kinetics
From 2014.igem.org
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Contents
Development of HPLC protocol to quantify TNT concentration
Figure 1: This animation shows three compounds in a sample; all of which have overlapping UV absorbance spectra. HPLC is able to quantify each compound present by separating each compound by elution time before measuring absorbance. This explains why a simple spectrophotometer test is inappropriate for complex mixtures such as those in our NemA kinetics experiment. Summary
HPLC is a valuable analytical technique capable of quantifying both nitroglycerin and TNT at concentrations far lower than than the quantification limit of the Raman spectrometer we had access to. This experiment shows the performance of the HPLC protocol we developed specifically to quantify TNT in a mixture containing the highly absorbent FMN cofactor of NemA. The TNT signal was successfully separated from that of FMN and a very precise, reliable and accurate standard curve was produced; shown in figure 3 of the results section.
Abstract
NemA is a protein that the iGEM Exeter team propose will allow E. coli to degrade nitroglycerin and TNT, at concentrations above those normally toxic to the cell. The team were unsuccessful in quantifying TNT using Raman spectroscopy and were therefore unable to show the protein NemA catalysing the breakdown of this compound as proposed. TNT was found to be immiscible with pure water resulting in extremely variable peak intensities when measuring TNT/water samples. This was likely due to the TNT forming a layer on top of the water. Among many others proposed, NemA catalyses the reaction shown in figure 2. As shown in figure 1, HPLC is a technique capable of isolating the absorbance signal of a compound which may be masked by other compounds with similar UV absorbance spectra. Both FMN and TNT were shown to absorb light in the 230 nm region. As FMN was a cofactor of NemA in the kinetics experiment, it's strong absorbance masked the signal of TNT and prevented the use of a simple spectrophotometer for TNT quantification.
Figure 2: proposed degradation of TNT Various hydroxylamino derivatives may be produced, as well as ammonium ions which could be used as a nitrogen source by the E. coli for growth. The following experimental account describes the HPLC setup parameters we developed to detect and create a standard curve for TNT.
Results
Choice of analytical procedure
NADH is a cofactor in the conversion of TNT to X. This opens up the possibility to simply measuring the catalytic rate of TNT degradation by following change in the absorbance at 340nm. However, very early in the process we realised that TNT also produces a significant absorbance at 340nm, a fact that would complicate our analytical procedure. We therefore chose to use High Performance Liquid Chromatography (HPLC) analysis. As can be seen (Fig X), HPLC separates TNT and NAD by elution time meaning that the overlap in absorbance values are not an issue.
TNT Standard Curve
A standard curve of TNT concentration was successfully determined. Integration of the area below the absorbance peak of TNT at the concentrations described, resulted in the standard curve shown in figure 3. For further details see materials and methods.
Figure 3: Standard curve demonstrating HPLC peak area response to different concentrations of TNT 
Figure 4: 3D HPLC absorbance graph showing the TNT standard curve. The solvent front and varying nitroglycerin absorbance are both clearly visible. Materials and Methods
Detection of TNT, NemA and its cofactors
Pure samples of FMN, NADH and NemA were injected into an Agilent Technologies infinity reverse phase high performance liquid chromatograph C18 column and their absorbance peaks identified by comparison with blanks (1:1 water, acetonitrile). Preliminary experiments showed a TNT absorption maximum at 230 nm. The elution time for the TNT peak was reliably situated at 0.8 minutes in all runs using the mobile phase gradient shown in figure 5.
Figure 5: HPLC mobile phase composition over the course of the run This mobile phase profile with a flow rate of 0.4 ml min-1 as well as an injection volume of 20 μL (minimum available) was kept constant over the course of the experiment to maintain elution profile consistency. A disturbance in the force of liquid applied to the column was shown to affect elution profiles to such an extent that identification and quantification of peak areas was unreliable. Buffer A consisted of 95% water and 5% acetonitrile whilst buffer B consisted of 95% acetonitrile and 5% water; both HPLC grade. 100% acetonitrile was not used to prevent polymerisation and build up of acetonitrile in the pumps. Initial runs consisting of NADH, FMN and NemA (as individual samples) dissolved in a 1:1 solution of acetonitrile and water showed no retention. The presence of these compounds was confirmed by the appearance of new absorbance peaks in the solvent front; previously absent in blank samples of a 1:1 solution of acetonitrile and water; shown in figure X. The mobile phase composition, shown in figure 5, allowed sufficient separation of the absorbance peak of TNT from the broad peaks of the highly absorbent peptides and cofactors in the solvent front; necessary for accurate and precise integration of the TNT peak area.
Quantification of TNT concentration
50 μL of stock TNT (0.44 M) in acetonitrile was diluted up to a total volume of 50 mL, with HPLC grade acetonitrile and water (1:1) to produce a 4.4 mM TNT solution. Serial dilutions of these solutions were performed to achieve the concentrations shown in figure 6.
Tube No. 1 2 3 4 5 6 7 8 9 10 [TNT] /mM 4.4 2.2 1.1 0.55 0.275 0.138 0.069 0.034 0.017 0 Figure 6 The absorbances of samples of these concentrations were measured using the HPLC technique described in figure 8.
Purification of His-tagged NemA
A 15 mL overnight culture of Exeter iGEM Team's construct 003 E. coli (expressing his-tagged NemA) was grown in LB over 12 hours from glycerol stock. A 10 mL sample of this overnight culture was added to 500 mL LB and left for 3 hours, whereupon the optical density of the culture reached 0.7 and 0.5 mL IPTG was added. 4 hours later the culture was centrifuged at 4500G for 20 minutes. The resulting pellet was resuspended in tris buffer and sonicated. The whole cell lysate was centrifuged for a further 30 minutes at 4500G. Purified NemA was separated from the supernatent by passing through a Nickel column.
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