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Team:Exeter/invivo
From 2014.igem.org
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Observing Degradation Products
Introduction
Aim
The aim of our experiment was to quantify the rate at which the aromatic ring of TNT is degraded using the change in sample colour, when TNT is mixed with constructs XenB and NemA. Top 10 will be used as a standard as it does not have amplified NemA or XenB TNT degradation pathways.
TNT Degradation
During the NemA and XenB-catalysed degradation of TNT, a series of nitrite groups as well aromatic ring reduction leads to formation of amino-dimethyl-tetranitrobiphenyl. During this process a hydride-Meisenheimer complex metabolite is formed. This degradation product has a distinct dark-brown colour [Vorbeck et.al 1994]. This degradation product causes reaction mixtures with XenB or NemA, mixed with TNT, to change from colourless to red, then to yellow [Pak 2000]. The resulting yellow colour results from four other degradation product which accumulate following aromatic ring reduction.
Results
In order to validate that our biobrick constructs were working as expected we ran a series of experiments to measure the degradation of TNT in vivo using our NemA and XenB constructs. In order to see the degradation rates over set periods of time one main reaction was left to run for 5 hours while samples were removed and either flash freezed using liquid nitrogen or placed in trichloroacetic acid to stop the enzymatic reaction and calculate the volume of TNT present at that time. We repeated this experiment (degradation experiment 2) in order to validate that the change was repeatable and to also narrow the time in which the degradation products were formed.
Experiment 1
Degradation experiment 1: 80 minutes after addition of 0.2ml of TNT to 0.4ml of overnight culture containing constructs, NemA had produced a dark red-brown colour. XenB showed a darker hue, whilst Top 10 control had not changed colour. The 0.4ml of overnight culture, made with 10ml of lysogeny broth (LB) and 0.01ml of chloramphenicol (CAM) antibiotic and construct glycerol stocks of XenB (001) and NemA (003) with control (Top 10). 
Degradation experiment 1:160 minutes after addition of TNT to 0.4ml of overnight culture, NemA and XenB had both formed a yellow colour whilst Top 10 control had not changed colour. This suggests that the rate of TNT aromatic ring reduction was greatly accelerated compared to Experiment 2
Degradation experiment 2 NemA:80 minutes after addition of TNT to 0.4ml of overnight culture, NemA had formed a dark-red brown colour. 
Degradation experiment 2 XenB:80 minutes after addition of TNT to 0.4ml of overnight culture, XenB had formed a slight brown colour 
Degradation experiment 2 Top10:80 minutes after addition of TNT to 0.4ml of overnight culture, Top 10 had not changed colour 
Degradation experiment 2 NemA:40 minutes after addition of TNT to 0.4ml of overnight culture, NemA had formed a slight brown colour 
Degradation experiment 2 XenB:40 minutes after addition of TNT to 0.4ml of overnight culture, XenB had formed a slight brown colour 
Degradation experiment 2 Top 10:40 minutes after addition of TNT to 0.4ml of overnight culture, Top 10 had not changed colour Methods
References
- Vorbeck, Claudia; Lenke, Hiltrud; Fischer, Peter; Hans-Joachim, Knackmuss (1994) Identification of a Hydride-MeisenheimerComplex as a Metabolite of 2,4,6-Trinitrotoluene by a Mycobacterium Strain ; Journal of Bacteriology
- Jeong W. Pak; Kyle L. Knoke; Daniel R. Noguera; Brian G. Fox; Glenn H. Chambliss (2000) Transformation of 2,4,6-Trinitrotoluene by Purified Xenobiotic Reductase B from Pseudomonas fluorescens I-C; Applied and Environmental Microbiology
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