Difference between revisions of "Part:BBa K1398003"
 
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<partinfo>BBa_K1398003 short</partinfo>
 
<partinfo>BBa_K1398003 short</partinfo>
  
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A construct created to degraded TNT and nitroglycerin.
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A construct created by the 2014 Exeter iGEM team to degrade TNT and nitroglycerin.
 
The construct contains the coding sequence for NemA (BBa_K1398002), an enzyme involved in the degradation of toxic compounds for their reuse in nitrogen metabolism.
 
The construct contains the coding sequence for NemA (BBa_K1398002), an enzyme involved in the degradation of toxic compounds for their reuse in nitrogen metabolism.
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The construct also contains a Lactose-inducible promoter (BBa_R0010 ), a strong RBS (BBa_B0034) and a double terminator made up of BBa_B0010 and BBa_B0012).
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The construct also contains a Lactose-inducible promoter (BBa_R0010 ), a strong RBS (BBa_B0034) and a double terminator made up of BBa_B0010 and BBa_B0012.
 
The protein has been codon-optimised for expression in E. coli.
 
The protein has been codon-optimised for expression in E. coli.
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<b>Exeter iGEM 2014</b>
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The Exeter 2014 team used Raman spectroscopy to show the breakdown of Nitroglycerin by NemA. The image below shows negative controls proving that NemA cannot perform this catalytic function without cofactors: FMN, NADH and NADPH. The conditions for this activity were room temperature and pressure and a physiologically relevant pH of 7.
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The minus protein control is greater than 100% of the starting concentration. This is likely a measurement error whereby the solvent has evaporated whilst focusing the Raman laser increasing the concentration of the compound of interest.
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https://static.igem.org/mediawiki/parts/8/82/Exeter2014_NemA_invitro.png
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The graph below shows how initial reaction velocity varies with initial concentration of the nitroglycerin in each reaction mixture. The concentrations of substrates and NemA are kept constant.
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https://static.igem.org/mediawiki/2014/6/67/NemA_degradation_of_nitroglycerin_kinetics_exp.png
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From these data a Hanes-Woolf plot was produced and allowed the Vmax and Km values to be determined: 6 mM and 21 umol per mg per min respectively.
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https://static.igem.org/mediawiki/2014/f/f8/Hanes.png
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Full article at: http://2014.igem.org/Team:Exeter/EnzymeValidation
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 19:21, 2 November 2014

NemA (Inducible Construct)

A construct created by the 2014 Exeter iGEM team to degrade TNT and nitroglycerin. The construct contains the coding sequence for NemA (BBa_K1398002), an enzyme involved in the degradation of toxic compounds for their reuse in nitrogen metabolism. The construct also contains a Lactose-inducible promoter (BBa_R0010 ), a strong RBS (BBa_B0034) and a double terminator made up of BBa_B0010 and BBa_B0012. The protein has been codon-optimised for expression in E. coli.

Exeter iGEM 2014

The Exeter 2014 team used Raman spectroscopy to show the breakdown of Nitroglycerin by NemA. The image below shows negative controls proving that NemA cannot perform this catalytic function without cofactors: FMN, NADH and NADPH. The conditions for this activity were room temperature and pressure and a physiologically relevant pH of 7.

The minus protein control is greater than 100% of the starting concentration. This is likely a measurement error whereby the solvent has evaporated whilst focusing the Raman laser increasing the concentration of the compound of interest.

Exeter2014_NemA_invitro.png

The graph below shows how initial reaction velocity varies with initial concentration of the nitroglycerin in each reaction mixture. The concentrations of substrates and NemA are kept constant.

NemA_degradation_of_nitroglycerin_kinetics_exp.png

From these data a Hanes-Woolf plot was produced and allowed the Vmax and Km values to be determined: 6 mM and 21 umol per mg per min respectively.

Hanes.png

Full article at: http://2014.igem.org/Team:Exeter/EnzymeValidation

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 746
    Illegal AgeI site found at 583
  • 1000
    COMPATIBLE WITH RFC[1000]