Difference between revisions of "Part:BBa K215090"

(Usage and Biology)
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===Usage and Biology===
 
===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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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.
  
  

Revision as of 01:53, 22 October 2010

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 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.


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]