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Team:Exeter/invivo
From 2014.igem.org
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Contents
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.
During the NemA and XenB-catalysed degradation of TNT, a series of nitrite group and aromatic ring reductions lead to formation of amino-dimethyl-tetranitrobiphenyl. During this process, a hydride-Meisenheimer complex degradation product 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 products, which accumulate following aromatic ring reduction. Therefore, the presence of a dark-brown colour within the construct and TNT mixture is a reliable indicator that the aromatic ring of TNT has been reduced.
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 4 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 TNT concentration within the the mixture formed was too low to be analysed by HPLC . When centrifuged, the resulting supernatant did not have a colour change. Raman could not be used as the TNT/water mixture is immiscible and forms a layer on top of fluid that is extremely variable in inelastic scattering feedback.
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. Experiment 2
Degradation experiment 2 Top 10: 40 minutes after addition of TNT to 0.4ml of overnight culture, Top 10 had not changed 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 NemA: 40 minutes after addition of TNT to 0.4ml of overnight culture, NemA had formed a slight brown colour. 
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. 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. These were then stored in the shaking incubator at 37 degrees and 300 rpm overnight.
Optical density for each of our overnight samples was measure using cuvettes and a light spectrometer. The following day we removed the overnight cultures from the incubator and measured the optical density. The light spectrometer could only accurately measure with readings less than one. Therefore we diluted the 1ml cuvette sample, by adding 0.1ml of overnight culture to 0.9ml of LB.
We removed 0.4ml of culture from the overnight cultures and placed them into labelled 2ml Eppendorfs. We then added 0.02ml of aqueous TNT into each Eppendorf and inverted in order to mix. So as to accurately sample the degradation product formation at select time intervals, 0.02ml was removed from each sample and placed in a new Eppendorf tube. These newly labelled Eppendorfs were then put into liquid nitrogen in order to snap freeze the samples at that reaction state. Once frozen, the samples were placed into a minus 80 oC freezer in order to maintain de-activation of the enzymes and prevent natural degradation of TNT. This removal and freezing process was repeated every 20 minutes for 4 hours.
References
- Vorbeck, Claudia; Lenke, Hiltrud; Fischer, Peter; Hans-Joachim, Knackmuss (1994) Identification of a Hydride-Meisenheimer Complex 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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