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.

Figure 1: 0.8% agarose gel containing the cut and uncut pET28-NPM1 vector'

This gel showed us we had successfully digested our plasmid, and it was ready for T4 ligation as well as transformation of the final cloning vector. To confirm the success of our transformation we used LB Agar plates containing Kanamycin which selected for the E. coli that had taken up the plasmid containing the kanamycin resistance gene shown in Figure 3.

Figure 2: pET28-NPM1 plasmid map"

Figure 3: Kanamycin plates used to confirm the presence of transformed TOP10 E. coli cells. a) Small BiT plate, b) Large BiT plate, c) Encapsulin plate."

It was clear from the plates shown in Figure 3 that we had been unsuccessful in our restriction digest, and that our colonies did not take up any of the plasmids. We attributed this to the restriction enzymes being used, as they were relatively old and inefficient. In response to this, we attempted a different transformation method using NEBuilder.

Primer Design

Prior to the second transformation, we designed forward and reverse primers for the pET28a vector and each insert which would be used to form our second plasmid via homologous recombination. These primers were created in SnapGene, described in our engineering section.