Difference between revisions of "Part:BBa K215090"
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===Usage and Biology=== | ===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 | + | 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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image:OpdA_zoom.png | image:OpdA_zoom.png | ||
</gallery> | </gallery> | ||
| − | The substrate vs. velocity curve above plots the rate of | + | 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-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> |
kcat (s-1): 17.6 <br> | kcat (s-1): 17.6 <br> | ||
Km (mM): 0.011 <br> | Km (mM): 0.011 <br> | ||
Revision as of 05:26, 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, 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-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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 994
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 91
Illegal AgeI site found at 286
Illegal AgeI site found at 625 - 1000COMPATIBLE WITH RFC[1000]