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Team:Exeter/invivo
From 2014.igem.org
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Observing Degradation Products
Summary
In this experiment we demonstrate that our NemA and XenB constructs are both capable of degrading the aromatic ring of TNT forming distinctive degradation products that result in colour change of the sample.
Aim
The aim of our experiment was to qualify the breakdown of 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. The mixture formed was unfortunately to complex to be analysed by HPLC and when centrifuged the resulting supernatant did not have a colour change. Raman could also not be used as the TNT and water mixture is imiscible as it forms an impenetrable layer that the laser cannot get through.
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
In order to test our constructs in vivo we first had to create cultures with an equal optical density in order that the results produced would be representative of the capability and rate of each enzyme and not the volume of cells. To do this we grew overnight cultures of constructs made with 10ml of lysogeny broth (LB) and 0.01ml of chloramphenicol (CAM) antibiotic and construct glycerol stocks of XenB (001) and NemA (003). The control, Top 10, did not contain any antibiotic. As a final control only LB was placed in the fourth falcon tube.
- Place 10 ml of LB into 4 Falcon tubes, labelled with 001, 003, LB and Top 10
- Put 0.1µl of CAM antibiotic into 001 and 003 falcon tubes. Do NOT put antibiotic into the top 10 LB. Leave just LB in the final tube.
- Using pipette tip scrape the tops of all three glycerol stocks (in -800c freezer) and place the pipette tip into each appropriate falcon tube
- Place these in the rotating incubator overnight
Day 2
Remove overnight cultures from incubator.
- Pipette 400uL of overnight culture from 001, 003 and Control (Top 10) into labelled 2mL Eppendorf tubes. Pipette 200uL of LB into final, labelled, 2ml Eppendorf. Do this under the fume hood.
- Pipette 20uL of aqueous TNT into each Eppendorf and shake to mix.
- Photograph the colour of each Eppendorf tube.
- Immediately pipette 20uL of the mixes into labelled 1.5 mL Eppendorfs. Snap freeze these samples in the liquid nitrogen flask. Store the samples in a freezer box.
- Put the 2mL Eppendorf tubes into the 37 degree rotating incubator.
- After half an hour snap freeze another 20ul sample in 1.5ml Eppendorf’s. Store the sample in the -80 freezer (In the freezer box).
- Take another sample after another half hour then take samples every hour for as long as you can.
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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