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

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.

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

Figure One:Amanda and Darren extract the frames for the experiment.

Prior to feeding the bees our constructs for the probeeotic, we had to ensure that E.coli could colonize the honeybee gut. E.coli harboring pSB1C3-RFP (red fluorescent protein or RFP expressing plasmid with chloramphenical or 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 separate 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.

Figure Two:Feeding the honeybees. The middle and left image display the cages in which the bees were kept in.

The positive results from the gut colonization experiment prompted testing resistance of bees to pesticides, such as imidacloprid and 6-chloronicitinic acid (6-CNA), after the gut is being colonized by E.coli strain capable of pesticides' degradation. 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. Subsequently, the bees with ingested E.coli were fed sucrose-water solution supplemented with either imidacloprid, 6-CNA, or no pesticide (as control). Bee death was recorded over four days and dead bees were analyzed for presence of ingested E. coli by plating homogenized bee gut on CM-plates. The presence of red colonies or colony PCR was applied to confirm the isolation of ingested E. coli.

Figure Three:The dissection for the alimentary canal of the honeybees.

Figure Four:The graphs above display the results of the resistance to imidacloprid experiments over two days.