Part:BBa_K5330019:Design
Results for UCNZ Team before Wiki Freeze
Overview The plan and desired result for this project was to design a proof-of-concept assay which could be used to detect the presence of the MAP-specific encapsulin 2A. This would come as a result of successful expression of wild type encapsulin, as well as two fusion proteins involving the encapsulin 2A monomers combined with Promega’s NanoBiT components Large BiT (herein LgBiT) and Small BiT (SmBiT). With these proteins expressed and purified, we could then mix the components together and observe a glow when LgBiT-encapsulin and SmBiT-encapsulin formed a cage, as the split luciferase subunits would form the NanoLuciferase and in the presence of NanoGlo substrate, would glow. As well as this, we aimed to understand our target protein and the cages it formed which was performed in the analytical ultracentrifuge, with the hypothesis being the formation of 60-mers.
Original Transformation The aim of our original DNA transformation was to linearise our pET28 plasmid vector using the restriction enzymes NcoI and XhoI and insert the genes for both the fusion proteins and the wild type encapsulin into the plasmid with the help of T4 Ligase to create our desired plasmids. The digested pET28 Vector were ran on a 0.8% agarose gel in TAE buffer shown in Figure 1. These plasmids could then be transformed into our TOP10 E. coli.
