Part:BBa_K215090

OpdA (phosphotriesterase)

OpdA is a phophotriesterase from Agrobacterium that can detoxify a broad range of organophosphate pesticides and nerve agents.

Usage and Biology

To test BBa _K215090 it was inserted into BBa_K215000 using standard biobrick assembly, resulting in https://parts.igem.org/Part:BBa_K215091 BBa_K215091]. OpdA was then produced and purified as described in the [http://2009.igem.org/Team:Washington/Notebook/IMAC_protocol UW 2009 iGEM team Notebook]. The purified protein was then tested for activity against paraoxon, for a detailed description of the assay please see the [http://2009.igem.org/Team:Washington/Project/Target#BioBricking_and_Characterization_of_OpdA 2009 UW iGEM wiki]. The resulting data is shown below.

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The substrate vs. velocity curve above plots the rate of paraoxon degradation (Vobs, y-axis) as a function of substrate concentration (x-axis). As observed in the curve above, at high substrate concentration this enzyme suffers from substrate inhibition, in the conditions it was assayed in. At lower concentrations it shows standard Michaelis-Menten kinetics, as depicted in the zoom in plot on the left. When this data was fit to a canonical substrate inhibition curve we obtained the following kinetic parameters:
kcat (s-1): 17.6
Km (mM): 0.011
Ksi (mM): 1.06
kcat/Km (M-1 s-1): 1.6 x 10⁶

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 994
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 91
  Illegal AgeI site found at 286
  Illegal AgeI site found at 625
  
• 1000
  COMPATIBLE WITH RFC[1000]