Team:British Columbia/Description

UBC iGEM 2015

 

Project Description

 

Bee populations around the world have been declining since the early 1990s. In 2015 alone, US beekeepers reported that 42% of their colonies died within the past year. Honeybee Colony Collapse Disorder (CCD) is a serious problem, given the ecological and economical importance of honeybees. CCD is a phenomenon in which adult worker bees die while away from the colony, leaving behind a non-functioning colony. Worker bees are usually the most severely affected, and bees that leave the hive are often unable to return, eventually resulting in the slow collapse of the hive.

Though the mechanisms by which CCD occurs are likely manifold and remain uncertain, neonicotinoid pesticides have been implicated. Imidacloprid, a neonicotinoid pesticide, is usually applied to plants as a seed coating. On germination and growth, imidacloprid is taken up by the plant and deposited in many plant tissues, poisoning all pests that then feed on the plant.

Death may occur as an acute effect at high doses, but this level of exposure to bees is not common in commercial agriculture. This fact is supported by the lack of (bee) cadavers surrounding a hive, where contaminated food stores would quickly decimate a population of bees.

Consequently, it is thought that the sublethal effects of imidacloprid are far more likely to lead to CCD. Sublethal chronic effects include a loss of coordination, lethargy, and lowered pathfinding abilities, severely impacting the efficacy of bee driven pollination.

We hypothesize that a strain of honeybee intestinal bacterium can be engineered to degrade imidacloprid, a widely-used neonicotinoid. In doing so, honeybees harboring the engineered bacterium will become resistant to common field doses of imidacloprid allowing for its sustained use while reducing the risk of CCD.

Gilliamella apicola is a bacterium native to the midgut of the bee. By engineering Gilliamella to metabolize imidacloprid into oxidizable organic compounds we can create a strain of Gilliamella capable of conferring resistance to imidacloprid. While the exact imidacloprid degradation pathway is unknown, an early step for degradation of neonicotinoids in vivo and in the environment involves the production of 6-chloronicotinic acid (6-CNA). Though 6-CNA requires a very high dose to induce acute toxicity, it still induces sublethal effects.

Click to view more on each subgroup.

In order to create our pro-bee-otic, bacteria specific to Apis mellifera were chosen: the β-proteobacteria, Snodgrassella alvi, and the γ-proteobacteria, Gilliamella apicola. By using microaerophilic bacteria that are unique to the honey bee gut, a specificity is acquired and the chances of pests acquiring the engineered imidacloprid resistant strains are decreased. However, due to the small amount of existing literature on G. apicola and S. alvi, an aspect of the project revolved around discovering methods of culturing the bacteria, inducing competence, and transforming them with a compatible plasmid.
Click here to read more.

Microbial strains able to degrade imidacloprid have been isolated from soil environments, however the specific microbial enzymes involved in the degradation pathway have not been characterized yet. Functional metagenomic approaches were designeded to screen large-insert environmental fosmid libraries obtained from Dr. Hallam lab for the imidacloprid-degrading phenotype. This approach does not depend on previous knowledge of enzymes involved in imidacloprid degradation and might target imidacloprid degrading enzymatic pathways which further can be incorporated in bee gut microorganisms.
Click here to read more.

Three cytochrome P450 enzymes (CYPs), CYP6CM1vQ, CYP6G1 and HUMCYPDB1, have been found to degrade imidacloprid into less toxic metabolites. In an attempt to synthesize E.coli, and further, G.apicola, to be able to degrade imidacloprid, we are constructing four vectors containing these genes. In addition to the CYP genes, each vector contains a pelB signal sequence and a AgCPR reductase to i) target the CYP to the inner membrane and ii) to recycle NADPH. Three vectors will contain one CYP gene and the fourth will contain all three CYP genes. In addition, we will optimize the genes for heterologous expression in E. coli through i) N-terminal truncations and/or ii) codon optimization.

Time permitting, we will optimize, demonstrate and characterize the in vivo detoxification of imidacloprid in E. coli through i) titrating cofactor or cofactor precursor concentrations (heme/heme precursors, NADPH), and ii) measuring detoxification kinetics during relevant growth and resting conditions. Click here to read more.

6-CNA is one of the byproducts of imidacloprid and is toxic, though to a lesser degree than imidacloprid. To degrade 6-CNA, it will be converted to a central metabolite in the TCA cycle, fumaric acid hence benefiting the host with the vector. In an attempt to synthesize E.coli, and further, G.apicola, to be able to degrade 6-CNA, CCH2, NIC C, NIC X, NIC D, NIC F and NIC E will be assembled into a vector via standard assembly. The vector contains each gene with a Ptac promoter, however only one of the genes will contain a Lac I repressor.

First the level of protein expression of CCH2 of the soluble and insoluble fraction at five temperatures (16 °C, 20 °C, 25 °C, 30 °C, 37 °C) will be tested to see the optimal temperature for expression and to check correct protein length. To test the degradation efficiency of CCH2, a resting cell assay as described in literature will be done. The quantity of 6-CNA will be validated via Gas chromatography- mass spectroscopy to determine how long degradation takes. To test degradation of 6-CNA, 6-CNA will be used as a sole carbon source with the assembled vector, the control without CCH2. Any growth by the vector with CCH2 plus the NIC cluster and no growth with the assembly missing CCH2 confirms that the pathway is functioning. Click here to read more.