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Revision as of 23:16, 26 August 2015
Project Description
According to the U.S. Department of Agriculture, bees pollinate 80% of our flowering crops, which constitute one third of everything we eat. From an economic standpoint, a study done at Cornell University estimates that honeybees pollinate $14 billion worth of seeds and crops per year in the United States alone. Unfortunately, global bee populations have been under attack since the early 1990s; in 2015, US beekeepers reported that 42% of their colonies died within the past year.
Honeybee Colony Collapse Disorder (CCD) refers to a phenomenon in which adult working bees disappear from the colony, leaving behind the queen bee and resulting in its eventual collapse. CCD remains a major concern across North America and Europe. Though the mechanisms by which CCD occurs are still unknown, neonicotinoids (a widely-used class of pesticides) and Nosema apis (an endoparasite that grows in the midgut of the honeybee following infection) have been implicated.
UBC’s 2015 iGEM team aims to create a strain of engineered honeybee intestinal bacterium capable of degrading the neonicotinoid pesticide imidacloprid, alongside an antifungal agent to eliminate N. apis. In doing so, we plan to render inoculated honeybees resistant to both Nosema and to common field doses of imidacloprid, allowing its sustained use while reducing the risk of CCD.
Gilliamella apicola is a bacterium that natively resides in the midgut of the bee. We believe that by engineering G. apicola to metabolize imidacloprid into harmless organic compounds as well as to produce gastrodianin, a potent antifungal agent, we can create a strain of G. apicola capable of conferring resistance to imidacloprid and Nosema, significantly reducing the risk of CCD once stably introduced into the bee gut.
Imidacloprid is known to be naturally degraded in the environment to 6-chloronicotinic acid (6-CNA). Though 6-CNA displays a significantly lower lethal dosage than imidacloprid, it remains bioactive to a small degree. As such, we plan to investigate downstream enzymes that further degrade 6-CNA, and use this novel pathway in G. apicola to degrade imidacloprid to a completely non-toxic product in the bee gut.
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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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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.
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.
UBC iGEM 2015