Difference between revisions of "Team:British Columbia/Description"
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| − | <p>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. | + | <p>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. Imidacloprid is a neurotoxin that binds irreversibly to acetylcholine receptors in the central nervous system, eventually leading to paralysis and death. |
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| − | <div class="tab-pane" id="b">Microbial strains able to degrade imidacloprid have been isolated from soil environments | + | <div class="tab-pane" id="b">Microbial strains able to degrade imidacloprid have been isolated from soil environments; however, the specific microbial enzymes involved in the degradation pathway have not yet been characterized. Functional metagenomic approaches were designed to screen large-insert environmental fosmid libraries obtained from the Hallam lab at UBC for an 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. <br /> Click <a href="https://2015.igem.org/Team:British_Columbia/Screening">here</a> to read more.</div> |
| − | <div class="tab-pane" id="c"><p>Imidacloprid is a neonicotinoid commonly used in pesticides around the world. Studies have shown that the use of this pesticide has adverse, and commonly fatal, effects on insects. Unfortunately, | + | <div class="tab-pane" id="c"><p>Imidacloprid is a neonicotinoid commonly used in pesticides around the world. Studies have shown that the use of this pesticide has adverse, and commonly fatal, effects on insects. Unfortunately, honeybees are also affected by imidacloprid. As such, imidacloprid has been cited as one of many factors contributing to colony collapse disorder (CCD). Previous work has been done to characterize enzymes capable of modifying imidacloprid into less toxic products. Out of all cited enzymes, three candidate enzymes were chosen due to their ability to modify imidacloprid and confer partial resistance to their hosts. Experiments were conducted to create bacterial strains containing the enzymes and to test their ability to modify imidacloprid into less toxic products. <br /> Click <a href="https://2015.igem.org/Team:British_Columbia/Imidacloprid">here</a> to read more.</p> |
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| − | <div class="tab-pane" id="d"><p>6-CNA is one of the byproducts of imidacloprid and is toxic, though to a lesser degree than imidacloprid. | + | <div class="tab-pane" id="d"><p>6-CNA is one of the byproducts of imidacloprid and is toxic, though to a lesser degree than imidacloprid. 6-CNA will be converted to a central metabolite in the TCA cycle, fumaric acid, which will benefit the host with the vector. In an attempt to synthesize <i>E.coli</i>, and further, <i>G.apicola</i>, to be able to degrade 6-CNA, CCH2, NIC C, NIC X, NIC D, NIC F and NIC E were assembled into a vector via standard assembly. The vector contains each gene with a Ptac promoter, as well as a Lac I repressor. </p> |
| − | <p>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) | + | <p>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) were tested to determine the optimal temperature for expression and to confirm correct protein length. To test the degradation efficiency of CCH2, a resting cell assay as described in literature was performed. The quantity of 6-CNA was validated via gas chromatography- mass spectroscopy to determine degradation rates. To test degradation of 6-CNA, 6-CNA was used as a sole carbon source. Any growth by the vector with CCH2 plus the NIC cluster and no growth with the assembly missing CCH2 would confirm that the pathway is functioning. <br /> Click <a href="https://2015.igem.org/Team:British_Columbia/6CNA">here</a> to read more.</p></div> |
<div class="tab-pane" id="e"><p>To test the viability of our 'pro-bee-otic', experiments colonizing honeybees gut with <i>E. coli</i> were conducted. The experiments first tested if bees fed a sugar solution containing <i>E. coli</i> would subsequently be colonized by <i>E. coli</i> in the gut. Secondly, experiments testing colonization of the bee gut with <i>E. coli</i> harboring pesticide degradation genes and resistance to the pesticide of interest. Bee gut colonization experiments were designed to mimic the real-life situation of how the 'pro-bee-otic' would be delivered to the bees through a sucrose-water solution and in a hive atmosphere. <br />Click <a href="https://2015.igem.org/Team:British_Columbia/Bee_Feeding_Prototype_Testing">here</a> to read more.</a> | <div class="tab-pane" id="e"><p>To test the viability of our 'pro-bee-otic', experiments colonizing honeybees gut with <i>E. coli</i> were conducted. The experiments first tested if bees fed a sugar solution containing <i>E. coli</i> would subsequently be colonized by <i>E. coli</i> in the gut. Secondly, experiments testing colonization of the bee gut with <i>E. coli</i> harboring pesticide degradation genes and resistance to the pesticide of interest. Bee gut colonization experiments were designed to mimic the real-life situation of how the 'pro-bee-otic' would be delivered to the bees through a sucrose-water solution and in a hive atmosphere. <br />Click <a href="https://2015.igem.org/Team:British_Columbia/Bee_Feeding_Prototype_Testing">here</a> to read more.</a> | ||
Revision as of 22:29, 18 September 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. Imidacloprid is a neurotoxin that binds irreversibly to acetylcholine receptors in the central nervous system, eventually leading to paralysis and death.
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.
Click here to read more.
Imidacloprid is a neonicotinoid commonly used in pesticides around the world. Studies have shown that the use of this pesticide has adverse, and commonly fatal, effects on insects. Unfortunately, honeybees are also affected by imidacloprid. As such, imidacloprid has been cited as one of many factors contributing to colony collapse disorder (CCD). Previous work has been done to characterize enzymes capable of modifying imidacloprid into less toxic products. Out of all cited enzymes, three candidate enzymes were chosen due to their ability to modify imidacloprid and confer partial resistance to their hosts. Experiments were conducted to create bacterial strains containing the enzymes and to test their ability to modify imidacloprid into less toxic products.
Click here to read more.
6-CNA is one of the byproducts of imidacloprid and is toxic, though to a lesser degree than imidacloprid. 6-CNA will be converted to a central metabolite in the TCA cycle, fumaric acid, which will benefit 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 were assembled into a vector via standard assembly. The vector contains each gene with a Ptac promoter, as well as 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) were tested to determine the optimal temperature for expression and to confirm correct protein length. To test the degradation efficiency of CCH2, a resting cell assay as described in literature was performed. The quantity of 6-CNA was validated via gas chromatography- mass spectroscopy to determine degradation rates. To test degradation of 6-CNA, 6-CNA was used as a sole carbon source. Any growth by the vector with CCH2 plus the NIC cluster and no growth with the assembly missing CCH2 would confirm that the pathway is functioning.
Click here to read more.
To test the viability of our 'pro-bee-otic', experiments colonizing honeybees gut with E. coli were conducted. The experiments first tested if bees fed a sugar solution containing E. coli would subsequently be colonized by E. coli in the gut. Secondly, experiments testing colonization of the bee gut with E. coli harboring pesticide degradation genes and resistance to the pesticide of interest. Bee gut colonization experiments were designed to mimic the real-life situation of how the 'pro-bee-otic' would be delivered to the bees through a sucrose-water solution and in a hive atmosphere.
Click here to read more.
UBC iGEM 2015