Our collection of parts contains genes required for modification of imidacloprid and degradation of 6-chloronicotinic acid (6-CNA). Final parts were assembled in pSB1C3 backbone harboring the chloramphenicol resistance gene and designed to have a LacI repressor, ribosome binding site, pTAC promoter, our degradation genes of interest, and double terminator.
The three composite parts each containing a cytochrome P450 (CYP) were designed to include an N-terminal pelB signal sequence to target expression to the periplasm and a cytochrome P450 reductase (CPR) for functionality of the CYP. The CPR was also made with it’s own designated rbs, promoter, pelB signal sequence, and terminator.
Composite parts with multiple degradation genes were designed so that each gene in the construct had a dedicated rbs, promoter, and double terminator.
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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, demonstrating that this part can be used to degrade 6-CNA to 6-HNA, a less toxic metabolite, was a big accomplishment (see data here). rem ipsum dolor sit amet, consectetur adipiscing elit. Sed posuere eget nulla a pharetra. Suspendisse ac venenatis odio. Sed blandit posuere erat a posuere. Suspendisse odio erat, elementum sodales ante vel, malesuada rutrum turpis.
This composite part consists of CYP6G1 is a cytochrome P450 (CYP) from Drosophila melanogaster that can modify imidacloprid to 4- and 5-hydroxyimidacloprid. These degradation products have been demonstrated to be less toxic to bees. Previous work by Middle East Technical University (METU) had worked on this part (
BBa_K1197013); however, it is not available from the registry. In attempts to make an improvement to this part, we included a pelB signal sequence immediately upstream of the gene to target CYP6G1 to the periplasm. Though we were not able to validate that this improved the part, previous literature on expression of CYP’s suggests that inclusion of a signal sequence greatly improves heterologous protein expression.
The objective of our probeeotic project was to genetically modify a native bee gut microbe to be capable of degrading imidacloprid and 6-CNA. A step towards that goal included generating a construct that consisted of multiple imidacloprid modification genes and genes for complete mineralization of 6-CNA to fumarate. Unfortunately, we were unable to generate any data on this composite part as E. coli harboring this plasmid did not grow well in liquid media (see data here).
Composite parts: Imidacloprid modification
BBa_K1813064 CYP6G1 from D. melanogaster with N-terminal PelB signal sequence and Musca domestica (house fly) cytochrome P450 reductase. CYP6G1 can convert imidacloprid to 4- and 5-hydroxy imidacloprid.
BBa_K1813065 CYP6CM1 from Bemisia tabaci with N-terminal PelB signal sequence and Musca domestica (house fly) cytochrome P450 reductase. CYP6CM1 can convert imidacloprid to 5-hydroxy imidacloprid.
BBa_K1813066 CYP2D6 from human with N-terminal PelB signal sequence and Musca domestica (house fly) cytochrome P450 reductase. CYP6G1 can convert imidacloprid to nitrosoimine derivative.
