Difference between revisions of "Team:British Columbia/Bee Feeding Prototype Testing"

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Revision as of 22:58, 18 September 2015

UBC iGEM 2015

 

Prototype

 

In order to test the viability of our pro-bee-otic, experiments on honeybees gut colonization with E.coli 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. 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.

Probeeotic Prototype Testing Details

The honeybees were generously donated from Dr. Leonard Foster at UBC and the techniques used below were taught by Amanda Van Haga, PhD candidate.

Prior to feeding the bees our constructs for the pro-bee-otic, we had to ensure that E.coli could colonize the honeybee gut. E.coli harboring pSB1C3-RFP (red fluorescent protein (RFP) expressing plasmid with chloramphenical(CM) resistance) was used as a molecular marker to test colonization. Bees were fed a 50% sucrose-water solution containing E.coli and bees were dissected to isolate the bee gut alimentary tract two and four days later. Homogenized bee gut was plated on CM-containing plates to select resistant E.coli fed to the bees from the native gut microbiota. Colonies expressing RFP could also then be visually identified to confirm the colonization by E.coli strain previously ingested by the bees. Click here to view the notebook which covers the experiment and results.




The positive results from the gut colonization experiment prompted testing resistance of bees to pesticides, imidacloprid and 6-chloronicitinic acid (6-CNA), after being fed E. coli capable of degrading these compounds. The honeybees were fed varying concentrations of E. coli harboring either the degradation genes or the RFP-containing plasmid for two days to allow colonization of the bee gut. Bees were subsequently fed the cells were fed sucrose-water solution supplemented with either imidacloprid, 6-CNA, or no pesticide. Bee death was recorded over four days and dead bees were analyzed for presence of ingested E. coli by plating homogenized bee gut on chloramphenicol. The presence of red colonies or colony PCR to detect presence of degradation genes was used to confirm isolation of ingested E. coli.