Coding

Part:BBa_K215090

Designed by: Chris Eiben   Group: iGEM09_Washington   (2009-09-30)
Revision as of 23:24, 21 October 2010 by Wusean (Talk | contribs) (Usage and Biology)

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.


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]


[edit]
Categories
Parameters
n/aOpdA (phosphotriesterase)