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Team:Exeter/EnzymeValidation
From 2014.igem.org
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Validation of Enzyme Function and Activity
Summary
This experiment shows that Raman spectroscopy can be used to measure the concentration of NG in aqueous solutions. It was shown to provide accurate quantifiable measurements of the concentration of NG where the main source of error was in the preparation of the samples themselves.
However the use of Raman spectroscopy to measure low concentrations of artificial compounds in the presence of organic material is not recommended. The issue with measuring concentrations in the presence of cells is the large amount of different systems inside the cell, these produced a large fingerprint region (800cm$^{-1}$ to 1800cm$^{-1}$) that can mask the peaks of the artificial compound. The cells had a large auto-florescence which contributed to the difficulty of extracting useful information from the spectrum.
Raman spectroscopy is better suited to measuring concentrations of nitroglycerin and TNT in vitro where higher concentrations can be used and auto-florescence can be kept to a minimum. For measurements in vivo the accuracy and rate of gathering data could be greatly improved by adding an nonreactive marker compound. This compound would have a known peak area and concentration and would allow for the calibration of other peaks present in a spectrum. This method could remove uncertainties in focusing the laser.
NemA is capable of catalysing the conversion of Nitroglycerin to various products using NADH and FMN as cofactors. The binding affinity of the protein for this substrate (the Michealis Menten constant, Km) and the maximum reaction velocity (Vmax), were determined and are comparable to the published values; shown in figure 1. NemA is therefore a suitable enzyme for use in our system and has been shown to function at the physiologically relevant pH of 7.
Abstract
XenB is a protein that the Exeter iGEM team propose will allow E.coli to degrade TNT and Nitroglycerin, at concentrations above those normally toxic to the cell. Among many others proposed, NemA catalyses the reaction shown in figure 2.
The aim of this experiment was to measure the degradation rate of trinitrotoluene (TNT) and nitroglycerin (NG) in vitro by pure samples of XenB.
Raman SpectroscopyThis is a technique used to observe vibrational, rotational and other low frequency modes in a system. It utilizes the inelastic scattering of monochromatic light from these low frequency modes by detecting the shift in reflected photon energy. Inelastic scattering occurs where part of the photons energy is transferred to the vibrational state resulting in a lower energy reflected photon. This shift can be used to give information about the types of bonds in the system and how many of these are present.
Typically the inelastically reflected light from the low frequency modes in a system is very weak in comparison to the elastically scattered light from the sample. In the past this had been the major barrier to the wider use of Raman spectroscopy. With recent improvements to band-stop filters the ease of processing the signal from the sample has been greatly improved and as a result Raman spectroscopy has been put to greater use.
Raman spectroscopy was used for this experiment because of its ability to both detect the compounds of interest and give quantifiable values for their concentration. In addition, this experiment will provide an indication as to whether this type of spectroscopy is suitable for use on aqueous organic samples.
Results, Analysis and Discussion
Choice of analytical procedureNADH is a cofactor in the breakdown of TNT and nitroglycerin by NemA. This opens up the possibility of simply measuring the catalytic rate of nitroglycerin or TNT degradation by following the change in absorbance at 340nm. However, very early in the process we realised that both TNT and nitroglycerin also produce a significant absorbance at 340nm; a fact that would complicate our analytical procedure. Raman spectroscopy was hypothesised to be capable of isolating the characteristic signals of nitroglycerin and TNT from a complex mixture, allowing quantification of these compounds.
The Raman Spectrum of TNT and NitroglycerinFigure 2 shows the Raman spectrum of NG and TNT observed using undiluted samples. The NG spectrum agrees with literature spectrum having two high intensity peaks at 800cm$^{-1}$ and 840cm$^{-1}$ [3]. The TNT spectrum corresponds to the literature spectrum [2] with its highest intensity peaks at 820$^{-1}$ and 1360 cm$^{-1}$.
The TNT spectrum has much lower intensity peaks compared to that of nitroglycerin. This is to be expected as the TNT sample was at a lower concentration (1mgml$^{-1}$) compared to that of Nitroglycerin (2mgml$^{-1}$).
Figure 2: The Raman spectrum of TNT and nitroglycerin. The TNT spectrum agrees with literature values having high intensity peaks at 820cm$^{-1}$ and 1360cm$^{-1}$ [2]. Nitroglycerin also agrees with literature values having two high intensity peaks at 800cm$^{-1}$ and 840cm$^{-1}$[3]. Figure 3 shows the normalized Raman spectrum for nitroglycerin at various concentrations between the wavenumbers of 850 cm$^{-1}$ and 750 cm$^{-1}$. The spectrum was normalized between 1200 cm$^{-1}$ and 600 cm$^{-1}$ by dividing by the average intensity between these points. The concentration is proportional to the peak area.
Figure 3: The normalized Raman spectrum for nitroglycerin. The concentration of nitroglycerin is proportional to the peak area. The figure 3 also shows the predicted background intensity level without the peaks. This was made by plotting a least square fitted line through measurements around the peak sites. The background intensity level increases with lower intensity peaks, this is an artifact of normalization.
Using the predicted background intensity the peak area, and thereby the concentration, can be calculated using the trapezium rule,
\begin{equation} \text{Area} = \sum_i^{N-1} \frac{I_i+I_{i+1}}{2}(k_{i+1}-k_i). \end{equation}Where $I_i$ is the intensity of the $i$th measurement and $k_i$ is the wavenumber of the $i$th measurement.
The peak area is plotted against the concentration of the prepared sample in figure 4. A least square fitted line (Red dashed line) does not intercept the origin of the graph. This error could be due to a systematic error in transferring the nitroglycerin into the dilutions. The stock nitroglycerin solution was quite viscous meaning that some extra nitroglycerin stuck to the outside of the pipette, this would have increased the amount of compound in each dilution.
The error in the NG concentration attempted to account for the difficulty of mixing the correct quantities of NG and water by estimating the quantity of nitroglycerin to be $\pm$ 0.5 $\mu$l. This is probably incorrect as the actual error would be biased towards greater concentrations of nitroglycerin.
Assuming that the peak height must be zero at zero concentration another least square fitted line was plotted, this time fixing the intercept to zero. This line will be used for determining concentrations of nitroglycerin in future measurements.
Figure 4: Peak area of Raman spectrum for known concentrations of nitroglycerin. The nitroglycerin concentrations are probably lower than the real values due to a systematic error incurred by the viscosity of the stock nitroglycerin solution. The error in the peak area is small, it is calculated via,
\begin{equation} \delta \text{Area} = \sigma \langle k_{i+1}-k_i \rangle \sqrt{\frac{N}{2}}, \end{equation}where $\sigma$ is the standard deviation from the predicted background intensity of the background intensity measurements. $\langle k_{i+1}-k_i \rangle$ is the average wavenumber gap between measurements and $N$ is the number of measurements. The error in the peak area diverges from the measured area considerably for the large numbers of measurements used to calculate it.
No standard curve for TNT was made. TNT was found to be immiscible with 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, when part this layer was deposited onto the slide the concentration would be unexpectedly high. However for most measurements only the water part of the sample would be placed onto the slide resulting in no peaks.
Due to the immiscibility of TNT and water, measurements of TNT concentration could not be made using Raman spectroscopy. Please see our high performance liquid chromatography page for a proposed solution to this problem.
Determination of NemA activityFigure 5 shows NemA reaction kinetics as a function of initial velocity, Vi, against initial TNT concentration (derived from taking a tangent at the steepest section of each series in figure 4). From this it can be concluded that the Vmax is...
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