Difference between revisions of "Team:British Columbia/Composite Part"

Cch2 is a novel chlorohydrolase that was identified in a soil bacterium, Bradyrhizobiaceae strain SG-6C, that is capable of dechlorinating 6-chloronicotinic acid (6-CNA) to 6-hydroxynicotinic acid (6-HNA). 6-CNA has been found to accumulate in soils and plant material following application of imidacloprid. Due to the associated toxicity of 6-CNA to bees, we designed a construct with cch2 in hopes that heterologous expression in a bee gut microbe, could provide the bee with resistance to the toxic effects of 6-CNA.

The cch2 construct was designed with a ribosome binding site, pTac promoter, and LacI repressor to allow for inducible expression. We confirmed expression of cch2 by SDS-PAGE and found maximal expression at 25°C. Furthermore, we demonstrated functionality of Cch2 using whole cell lysate to assay conversion of 6-CNA to 6-HNA. Analysis was performed using GC-MS. A time-course assay with E. coli harboring the cch2 construct was used to determine the conversion rate of 6-CNA to 6-HNA.

Validation of this part was critical to link the modification of imidacloprid by CYPs with the degradation pathway encoded by the nicCXDFE pathway, which takes 6-HNA to fumarate. Subsequently, fumarate can be used in the microorganism’s central metabolism.

Figure 1: Consumption of 6-CNA and appearance of 6-HNA during 4 hour Resting Cell Assay. Metabolites were identified by GC/MS. The control is E.Coli with an empty PSB 1C3 plasmid. Peaks representing 6-CNA and 6-HNA peaks were in agreement with standards run previously. Degradation of 6-CNA began nearly instantly as the time zero samples had presence of the 6-HNA peak, as seen on the graph. At 60 minutes, all of the 6-CNA had been degraded and only 6-HNA peaks appeared on the GC/MS chromatogram.