Team:UAlberta/Demonstrate

Demonstrate

Over the course of this iGEM season, with parallel efforts in both our bee lab and synbio lab to maximize our limited time, Team UAlberta was able to perform key experiments that together serve as a proof-of-principle demonstration that our system works, which we will recap here. For full experimental results, please proceed to the results section for the bee and synbio lab

Our solution to addressing N. ceranae infection is predicated on two hypotheses:

1. We can engineer E. coli to overexpress the enzymes needed to drive the production of PPIX.
2. PPIX is effective in reducing the spore load of N. ceranae

Engineering E. coli to overproduce PPIX production

To address the first hypothesis, we performed two experiments. Our first experiment involved overexpressing 5-aminolevulinic synthase, which has been previously shown to be sufficient to drive PPIX production, in DH10B to see if PPIX can be produced as the literature suggests [1]. We extracted porphyrins secreted in the media and characterized them via fluorescence spectroscopy and thin layer chromatography. The similarities of the fluorescence and TLC results between our stock (Enzo Life Sciences) and extracted porphyrins strongly suggest that overexpressing the enzyme encoded by HemA alone is sufficient to drive porphyrin production - specifically PPIX production.