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

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All constructs were made using restriction digest and ligation methods (standard assembly). pelB was inserted upstream of the CYPs and the AgCPR using megaprimer mutagenesis PCR. Please refer to “Complete Construct Table” for all construct designs. Refer to “Restriction Digest Protocol” for standard assembly methods. Further, refer to the “Mutagenesis PCR” protocol used to insert pelB upstream of the CYPs and the AgCPR.
 
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Revision as of 21:30, 18 September 2015

UBC iGEM 2015

 

Degradation of
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.


Goals

Three cytochrome P450’s, CYP6CM1, CYP6G1 and CYP2D6, have been found to modify imidacloprid (IMI) into less toxic compounds, primarily 4- and 5-hydroxyimidacloprid. Our project goals were to:

  • Optimize the CYP’s for heterologous expression in E. coli through codon optimization.
  • Create three E. coli strains, each expressing either CYP6CM1, CYP6G1 or CYP2D6 by using a BioBrick compatible expression system (pSCB1C3) to enable E. coli to modify imidacloprid.
  • Test for protein expression and characterize in vivo detoxification of imidacloprid in E. coli through SDS-PAGE and High Performance Liquid Chromatography (HPLC), respectively.



    Construct Development

    All constructs were made using restriction digest and ligation methods (standard assembly). pelB was inserted upstream of the CYPs and the AgCPR using megaprimer mutagenesis PCR. Please refer to “Complete Construct Table” for all construct designs. Refer to “Restriction Digest Protocol” for standard assembly methods. Further, refer to the “Mutagenesis PCR” protocol used to insert pelB upstream of the CYPs and the AgCPR.



    heading 4



    nicD