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.

Second Transformation

After successfully designing and amplifying the genes with the primers added, removing the methylated pET28a DNA with DpnI and constructing the plasmids with NEBuilder assembly, transformation was performed, using kanamycin to generate successfully transformed SHuffle T7 E.coli colonies (Figure 4).

Figure 4: Successful Transformed Colonies containing the plasmids with all the constructs: a) SmBit-Encapsulin fusion protein, b) Wild type Encapsulin c) LgBiT-Encapsulin fusion protein."

Expression Trials

The purpose of these expression trials was to understand at what conditions our proteins best expressed, to ensure a successful expression of the protein in future iterations. At a range of final IPTG concentrations (0.1mM, 0.5mM, 1.0mM) and temperatures (20°C, 30°C, 37°C), the transformed cells were incubated to allow for overexpression of our proteins, after which a whole cell and soluble SDS-PAGE (MES buffer, 200V for 22 minutes, stained with simply blue) was run for each protein with lanes containing the different conditions. For the following figures L is the ladder and U is the uninduced culture.

Figure 5: Protein gel of Soluble encapsulin protein showing clear signs that expression. Clear bands in lanes 2, 10, 12 and 14"

Figure 6: Protein gel contain whole cell lysate containing overexpressed wild type encapsulin."

Figure 7: Protein gel containing soluble LgBiT-encapsulin fusion protein. Clear bands in lanes 9, 10,12 and 14."

Figure 8: Protein gel containing whole cell lysate with overexpressed LgBiT-encapsulin."

Figure 9: Protein gel containing any overexpressed soluble SmBiT-encapsulin fusion proteins. No clear bands."

Figure 10: Protein gel containing whole cell lysate after SmBiT-encapsulin overexpression."

It was determined from the soluble SDS-PAGE gels that 37oC with 0.1mM IPTG was the most consistent expression conditions for all three of the proteins. This was the expression conditions that were used for all following protein expressions. As well as this, we knew that the proteins were also soluble, and we could continue as planned with soluble protein purification methods.

Protein Purification

Immobilised Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC): After the IMAC was performed on all three proteins by capturing the His-Tag of the proteins on the column and using the ӒKTA, they were concentrated down, and the concentration of protein was determined using the NanoDrop which were as follows:

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Protein	Concentration
Encapsulin	2.00mg/mL
LgBiT-Encapsulin	2.26mg/mL
SmBiT-Encapsulin	2.25mg/mL