Difference between revisions of "Team:British Columbia/Description"

 
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<p>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.</p>
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<img src="https://static.igem.org/mediawiki/2015/1/10/Flowchart_lab_overview.png"
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align="left"; width="500px"; style="padding-right:10px"><p align="justify">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.</p>
  
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<p>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) has been implicated.</p>
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<p align="justify">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.
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<p>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 <i>N. apis</i>. In doing so, we plan to render inoculated honeybees resistant to common field doses of imidacloprid, allowing its sustained use while reducing the risk of CCD.</p>
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<p align="justify">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.
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<p><i>Gilliamella apicola</i> is a bacterium that natively resides in the midgut of the bee. We believe that by engineering <i>G. apicola</i> to metabolize imidacloprid into harmless organic compounds as well as to produce gastrodianin, a potent antifungal agent, we can create a strain of <i>G. apicola</i> capable of conferring resistance to imidacloprid, significantly reducing the risk of CCD once stably introduced into the bee gut.</p>
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<p align="justify">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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<p>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 <i>G. apicola</i> to degrade imidacloprid to a completely non-toxic product in the bee gut.</p>
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<p align="justify">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.
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<p align="justify"><i>Gilliamella apicola</i> is a bacterium native to the midgut of the bee. By engineering <i>Gilliamella</i> to metabolize imidacloprid into oxidizable organic compounds we can create a strain of <i>Gilliamella</i> 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.</p>
  
  
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                     <li><a href="#a" data-toggle="tab"><strong>Growing the Bacteria</strong></a></li>
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                     <li><a href="#a" data-toggle="tab" style="text-indent:0;">Genetic Tool Development</a></li>
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<div class="tab-pane" id="a"><p>In order to create our pro-bee-otic, we chose the betaproteobacteria <i>Snodgrassella alvi</i> and the gammaproteobacteria <i>Gilliamella apicola</i>, both specific to <i>Apis mellifera</i>. 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 <i>G. apicola</i> and <i>S. alvi</i>, an aspect of our project revolved around discovering methods of culturing the bacteria, inducing competence, and transforming them with a compatible plasmid.</p>
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<div class="tab-pane" id="a"><p align="justify">In order to create our pro-bee-otic, bacteria specific to <i>Apis mellifera</i> were chosen: the β-proteobacteria, <i>Snodgrassella alvi</i>, and the  γ-proteobacteria, <i>Gilliamella apicola</i>. 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 <i>G. apicola</i> and <i>S. alvi</i>, an aspect of the project revolved around discovering methods of culturing the bacteria, inducing competence, and transforming them with a compatible plasmid.<br />Click <a href="https://2015.igem.org/Team:British_Columbia/Growing">here</a> to read more.</p></div>
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<p>To transform <i>G.apicola</i> and <i>S.alvi</i> with a plasmid, we have used heat shock and electroporation transformations and conjugation with S17 and SM10 <i>E.coli</i>. 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. </p><p>Click <a href="https://2015.igem.org/Team:British_Columbia/Growing">here</a> to read more.</p></div>
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<div class="tab-pane" id="b">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 <a href="https://2015.igem.org/Team:British_Columbia/Screening">here</a> to read more.</div>
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<div class="tab-pane" id="b"><p align="justify">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>
  
  
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<div class="tab-pane" id="c"><p>Three cytochrome P450 enzymes (CYPs), CYP6CM1vQ, CYP6G1 and HUMCYPDB1, have been found to degrade imidacloprid into less toxic metabolites. In an attempt to synthesize <i>E.coli</i>, and further, <i>G.apicola</i>, 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 <i>E. coli</i> through i) N-terminal truncations and/or ii) codon optimization.</p><p>Time permitting, we will optimize, demonstrate and characterize the in vivo detoxification of imidacloprid in <i>E. coli</i> through i) titrating cofactor or cofactor precursor concentrations (heme/heme precursors, NADPH), and ii) measuring detoxification kinetics during relevant growth and resting conditions. 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="c"><p align="justify">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>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 <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 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. </p>
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<div class="tab-pane" id="d"><p align="justify">6-chloronicotinic acid (6-CNA) is an intermediate in imidacloprid modification that is both toxic to bees and a persistent environmental contaminant. Our degradation pathway allows for the formation of a TCA cycle intermediate, fumaric acid, which can be utilized in central metabolism. To potentially aid in the increased survival of honey bees from the effects of imidacloprid, our probeeotic must be capable of not only modifying imidacloprid, but also detoxifying the 6-CNA breakdown product. This is achieved by degradation by <i>cch2</i> and the <i>nic</i> cluster. <i>cch2</i> has been isolated from <i>Bradyrhizobiaceae</i> SG-6C and the <i>nic</i> cluster has been isolated from <i>Pseudomonas putida</i> KT2440.  </p>
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<br /> Click <a href="https://2015.igem.org/Team:British_Columbia/6CNA">here</a> to read more.</p></div>
  
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<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) 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 <a href="https://2015.igem.org/Team:British_Columbia/6CNA">here</a> to read more.</p></div>
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<div class="tab-pane" id="e"><p align="justify">In order to test the viability of our probeeotic, experiments on honeybees gut colonization with <i>E. coli</i> containing pesticide-degrading genes were designed and conducted. First, the bee gut colonization was verified after feeding bees a sucrose-water solution supplemented with <i>E. coli</i>. Second, experiments testing colonization of the bee gut with <i>E. coli</i>, harboring pesticide-degradating genes were performed. Bee gut colonization experiments were designed to mimic the real-life situation of how the probeeotic would be delivered to the bees through a sucrose-water solution in the hive environment. <br />Click <a href="https://2015.igem.org/Team:British_Columbia/Design">here</a> to read more.</a>
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Latest revision as of 23:34, 3 October 2015

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. 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.

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.
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-chloronicotinic acid (6-CNA) is an intermediate in imidacloprid modification that is both toxic to bees and a persistent environmental contaminant. Our degradation pathway allows for the formation of a TCA cycle intermediate, fumaric acid, which can be utilized in central metabolism. To potentially aid in the increased survival of honey bees from the effects of imidacloprid, our probeeotic must be capable of not only modifying imidacloprid, but also detoxifying the 6-CNA breakdown product. This is achieved by degradation by cch2 and the nic cluster. cch2 has been isolated from Bradyrhizobiaceae SG-6C and the nic cluster has been isolated from Pseudomonas putida KT2440.


Click here to read more.

In order to test the viability of our probeeotic, experiments on honeybees gut colonization with E. coli containing pesticide-degrading genes were designed and conducted. First, the bee gut colonization was verified after feeding bees a sucrose-water solution supplemented with E. coli. Second, experiments testing colonization of the bee gut with E. coli, harboring pesticide-degradating genes were performed. Bee gut colonization experiments were designed to mimic the real-life situation of how the probeeotic would be delivered to the bees through a sucrose-water solution in the hive environment.
Click here to read more.