Difference between revisions of "Part:BBa K215090"
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OpdA is a phophotriesterase from Agrobacterium that can detoxify a broad range of organophosphate pesticides and nerve agents.
 
OpdA is a phophotriesterase from Agrobacterium that can detoxify a broad range of organophosphate pesticides and nerve agents.
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===Usage and Biology===
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To test BBa _K215090 it was inserted into BBa_K215000 using standard biobrick assembly.  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 paroxoan, 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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<gallery heights=400px widths=350>
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image:OpdA_full.png
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image:OpdA_zoom.png
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</gallery>
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The substrate vs. velocity curve above plots the rate of paroxoan 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-Menton 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:<br>
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kcat (s-1): 17.6 <br>
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Km (mM): 0.011 <br>
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Ksi (mM): 1.06  <br>
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kcat/Km (M-1 s-1):  1.6 x 10<sup>6</sup>
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Revision as of 01:11, 19 October 2009

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. 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 paroxoan, 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.


  • OpdA full.png
  • OpdA zoom.png

The substrate vs. velocity curve above plots the rate of paroxoan 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-Menton 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]