• Home
• The Problem
• Our Project
  • Modelling
  • The Enzymes
  • Enzyme Validation
  • in vivo: Raman
  • in vivo: Observations
  • HPLC
  • Kill Switches
  • Xenobiotic Tolerance
  • Detection of Xenobiotics
  • iLOV Characterisation
  • Our Parts
• The Team
  • The Undergraduate Team
  • Supervisors
  • Official Profile
  • Attributions
  • Sponsors
• Medal Requirements
• Notebook
  • Lab Book
  • Protocols
• Outreach
  • Beyond The Bench
  • Stakeholders
  • Presentations
  • ERASE-The Game!
• 

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

Experiment 2

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 C at 300 rpm overnight.

The next 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 sample removed by adding 0.1ml of overnight culture to 0.9ml of LB. Using the standard curve created through testing of TNT degradation using HPLC we knew that the ideal concentration for TNT detection was between 0.014-0.44mm. Therefore we added 0.1ml of TNT to our 10ml falcon tube of overnight culture.

References

Follow @ExeteriGEM2014