Difference between revisions of "Team:British Columbia/Imidacloprid"

Imidacloprid is a neonicotinoid commonly used in pesticides around the world [1]. Studies have shown that the use of this pesticide has adverse effects on insects since it acts as an neurotoxin by binding and blocking the nicotinergic neuronal pathway [2]. As a result, honey bees become paralyzed and eventually die [3]. Studies have shown that there are three cytochrome P450s (CYPs) capable of modifying imidacloprid into less toxic substances [4-6]. In light of such findings, experiments were conducted in order to demonstrate that CYP6G1, CYP2D6, and CYP6CM1 can modify imidacloprid into less toxic compounds. In order to test for such modifications, constructs were created to code for: 1) a cytochrome P450 (CYP), 2) a targeting signal sequence, pelB, and 3) an NADPH-cytochrome P450 oxidoreductase (CPR).

In an attempt to improve CYP expression, pelB was added to the constructs. pelB targets the CYPs to the periplasm of the cell, which is where the CYPs are fully functional [7]. In addition, a P450 oxidoreductase (CPR) is also required to make the CYPs fully functional. AgCPR, a P450 oxidoreductase from Anopheles gambiae, reduces the CYPs by transferring two electrons from NADPH, a reaction necessary for the hydroxylation of imidacloprid by the CYPs [8]. As such, AgCPR was included in the constructs.

Final constructs were inserted into E. coli and tests were conducted to test the expression of the CYPs in E. coli as well as to test the degradative ability of the CYPs on imidacloprid. Two methods were used in the construction of the final constructs: 1) megaprimer mutagenesis PCR to insert pelB and 2) standard assembly to assemble the CYPs and the AgCPR. Gene expression was tested via SDS-PAGE electrophoresis and imidacloprid metabolism assays were conducted using HPLC.

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