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Team:Exeter/invivo
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| - | <figcaption><b>Experiment 1:</b> 0.4ml of overnight culture | + | <figcaption><b>Experiment 1:</b> 2ml Eppendorf containing 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). 80 minutes after addition of 0.2ml of TNT NemA had produced a dark red-brown colour. XenB showed a darker hue, whilst Top 10 control had not changed colour. </figcaption> |
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Revision as of 21:45, 17 October 2014
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Measuring in vivo Degradation Using Metabolite Colour Change
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 colour when TNT is mixed with constructs 001 and 003. Top 10 will be used as a standard as it does not have amplified NemA or XenB TNT degradation pathways. A standard of LB will also be used as there should be no reaction between TNT and LB.
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 metabolite has a distinct dark-brown colour [Vorbeck et.al 1994]. This metabolite 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 metabolites which accumulate following aromatic ring reduction.
Results
Experiment 1: 2ml Eppendorf containing 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). 80 minutes after addition of 0.2ml of TNT NemA had produced a dark red-brown colour. XenB showed a darker hue, whilst Top 10 control had not changed colour. 
Figure 1: 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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