In order to create our pro-bee-otic, we chose the betaproteobacteria Snodgrassella alvi and the gammaproteobacteria Gilliamella apicola, both specific to Apis mellifera. By implementing our system in these microaerophiles which are native and unique to the honey bee gut, we are inhibiting other insects from acquiring the engineered, imidacloprid resistant strains. However, due to the small amount of existing literature on G. apicola and S. alvi, an aspect of our project revolved around discovering methods of culturing the bacteria, inducing competence, and transforming them with a compatible plasmid.

To transform G.apicola and S.alvi with a plasmid, we have used heat shock and electroporation transformations and conjugation with S17 and SM10 E.coli. We have attempted many gram-negative bacteria specific broad-host plasmids such as: RP1, PBBR1MCS-2, PBBR3, PBBR4, PIND4, PKT210, and PRK293. Upon transformation, we plan to insert a marker into the plasmid and feed the bees with our constructs.

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There is no described pathway that degrades imidacloprid to 6-CNA, though 6-CNA has been described as accumulating in soils and plants. In light of this, we are screening fosmid libraries for clones capable of imidacloprid degradation when grown on imidacloprid as sole C- or N-source. Specifically, we will screen for isolates able to use imidacloprid, and then determine the average imidacloprid toxicity levels for E. coli to screen for isolates that can tolerate higher concentrations. For high-throughput functional screening purposes, we will utilize pooled screening by combining 384 clones to reduce screening time and cost. After determining clone(s) of interest, we will full-length sequence the insert to determine potential degrading genes to complement our work engineering a pathway with known degradation genes. 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.

Degradation by the CYP’s results in the formation of 6-chloronicotionic acid (6-CNA), which will be degraded further by CCH2 and the Nic cluster. Due to 6-CNA being toxic, though to a lesser degree than imidacloprid, it must be further degraded. 6-CNA 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.