Team:Exeter/enzyme-kinetics

From 2014.igem.org

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<h2>Summary</h2>
<h2>Summary</h2>
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<p>
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<p>HPLC is a valuable analytical technique capable of quantifying both nitroglycerin and TNT at concentrations far lower than than the lower limit of quantification of the Raman spectrometer we had access to.</p>
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NemA and Xen B are capable of catalysing the conversion of TNT to various products using NADH and FMN as cofactors. The binding affinity of each 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 and XenB are therefore suitable enzymes for use in our system and have been shown to function at the physiologically relevant pH of 7.
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<table align="center" border="1">
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<td></td>
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<td>NemA Vmax (TNT)</td>
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<td>XenB Vmax (TNT)</td>
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<td>NemA Vmax (Nitroglycerin)</td>
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<td>XenB Vmax (Nitroglycerin)</td>
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<td>NemA Km (TNT)</td>
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<td>XenB Km (TNT)</td>
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<td>NemA Km (Nitroglycerin)</td>
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<td>XenB Km (Nitroglycerin)</td>
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<td>Experimental Results</td>
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<td>Published Values</td>
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<td>8</td>
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<td>15</td>
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<p align="center"><i>Figure 1</i></p>
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<h2>Abstract</h2>
<h2>Abstract</h2>
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<p>NemA and XenB are two proteins that the iGEM Exeter team propose will allow E.coli to degrade TNT, at concentrations above those normally toxic to the cell. Among many others proposed, NemA catalyses the reaction shown in figure 2.
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<p>NemA is a protein that the iGEM Exeter team propose will allow <i>E.coli</i> to degrade nitroglycerin and TNT, at concentrations above those normally toxic to the cell. Among many others proposed, NemA catalyses the reaction shown in figure 2.
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<p>
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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.  
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.  
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The following experimental account describes the protocol used to confirm the substrate target of NemA and XenB and analyse the respective kinetic capabilities of these enzymes.
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The following experimental account describes the HPLC setup parameters we developed to detect and create a standard curve for TNT.</p>
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</p>
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<h2>Results</h2>
<h2>Results</h2>
<h4>Choice of analytical procedure</h4>
<h4>Choice of analytical procedure</h4>
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<p>
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<p>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.</p>
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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.
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</p>
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<h4>TNT Standard Curve</h4>
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<b>TNT Standard Curve</b>
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<p>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.</p>
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A standard curve of TNT concentration was determined first. 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.
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<figure>
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<h4>Determination of NemA activity</h4>
 
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Purified NemA protein was assayed for its ability to degrade TNT at a range of concentrations: from 0 mM TNT up to 4.4 mM TNT. The reaction is a stopped enzyme assay. In this system the reaction is started by the addition of NemA to a reaction mix containing TNT, FMN and NADH. The reaction is stopped by the addition of hydrochloroacetic acid at predetermined intervals and the concentration of TNT is then assayed using the HPLC. These data are shown in figure 4. Full details can be seen in the materials and methods.
 
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<img class="large_centre_image" src="https://static.igem.org/mediawiki/2014/e/e9/TNT_conc_over_time.png" height="560" width="660" style="margin: 0px 250px opx 0px">
 
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<figcaption>Figure 4</figcaption>
 
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Figure 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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<img class="large_centre_image" src="https://static.igem.org/mediawiki/2014/1/15/NemA_initial_velocities.png" height="560" width="660" style="margin: 0px 250px opx 0px">
 
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<figcaption>Figure 5: NemA reaction kinetics as a function of initial velocity, Vi, against initial TNT concentration</figcaption>
 
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The data can either be plotted as a Lineweaver-Burke plot (figure 6), or as a Hanes plot (figure 7) to more accurately determine the Km values. From these it can be concluded that the Km of NemA for TNT substrate is...
 
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<h2>Materials and Methods</h2>
<h2>Materials and Methods</h2>
<h4>Detection of TNT, NemA and its cofactors</h4>
<h4>Detection of TNT, NemA and its cofactors</h4>
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<p>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).
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Pure samples 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).
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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 8.</p>
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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 8
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<h4>Quantification of TNT concentration</h4>
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<b>Quantification of TNT concentration</b>
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<p>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 9.</p>
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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 9.
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<table align="center" border="1">
<table align="center" border="1">
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<p align="center"><i>Figure 9</i></p>
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<figcaption>Figure 9</figcaption>
<p>The absorbances of samples of these concentrations were measured using the HPLC technique described in figure 8.</p>
<p>The absorbances of samples of these concentrations were measured using the HPLC technique described in figure 8.</p>
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<h4>Purification of His-tagged NemA</h4>
<h4>Purification of His-tagged NemA</h4>
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<p>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.</p>
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Insert brief description of how NemA was expressed in cells i.e. IPTG concentration, OD at induction etc.
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<h4>NemA Kinetic Analysis</h4>
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His tagged NemA was separated from whole cell lysate by running through a nickel column and were subsequently stored at 5oC. The resulting aqueous protein was quantified by absorbance in a Thermo Scientific Nanodrop 2000c using its published extinction coefficient of …
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A master mix of FMN (0.14 mM), NADH (0.14 mM) and NemA (x mM) was created. To samples of this master mix, TNT was added to create final reaction mixture concentrations of: 4.4, 2.2, 1.1, 0.55, 0.28, 0.14, 0.07, 0.03 and 0.02 mM. After incubation at 20oC, each reaction was stopped after 1, 5, 10 and 30 minutes with the addition of 20 µl of trichloroacetic acid (240 mg/mL). The resulting mixture was quick-frozen and stored at −70°C before HPLC analysis.  
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<h2> Navigation </h2>
<h2> Navigation </h2>

Revision as of 20:21, 16 October 2014

Exeter | ERASE

Kinetic Analysis of NemA and XenB by HPLC

(this will be figure X, a totally EPIC animation of a 2D absorbance graph revolving into a 3D HPLC absorbance graph to highlight the importance of having a 3rd axis for elution time)

Summary

HPLC is a valuable analytical technique capable of quantifying both nitroglycerin and TNT at concentrations far lower than than the lower limit of quantification of the Raman spectrometer we had access to.

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. Among many others proposed, NemA catalyses the reaction shown in figure 2.

Figure 2:

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

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 8.

Figure 8: 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 1, 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 9.

Tube No.12345678910
[TNT] /mM4.42.21.10.550.2750.1380.0690.0340.0170
Figure 9

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.

Navigation

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