Difference between revisions of "Part:BBa K215090"

Revision as of 14:21, 26 October 2017

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

Improvement

2017 SSTi-SZGD has improved the this part by add a TorA signal peptide to N-terminus of opdA gene and gave it a new name TorA-opdA(BBa_K2244003).

Sequence and Features