DNA

Part:BBa_K5330019:Design

Designed by: Isabel Bradley   Group: iGEM24_UCNZ   (2024-09-16)

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 we 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 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: Figure 2: pET28 plasmid map with the restriction sites in bold, NEBuilder primer locations in magenta and T7 promoter encircled

Figure 3: LB Agar plates with Kanamycin 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: 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: Figure 5: SDS-PAGE gel of Soluble Wild-Type Encapsulin protein showing clear signs that expression. Clear bands in lanes 2, 10, 12 and 14. Red box indicates expected size of protein band

Figure 6: Figure 6: SDS-PAGE gel of whole cell lysate containing overexpressed Wild-Type Encapsulin. Red box indicates expected size of protein band

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:

Protein Concentration
Encapsulin 2.00mg/mL
LgBiT-Encapsulin 2.26mg/mL
SmBiT-Encapsulin 2.25mg/mL
These concentrated samples were then run through the SEC column using the ӒKTA to further purify them, yielding the following concentrations:
Protein Concentration
Encapsulin 0.62mg/mL
LgBiT-Encapsulin 0.19mg/mL
SmBiT-Encapsulin 1.17mg/mL
Immobilised Metal Affinity Chromatography (IMAC) and Dialysis: Due to the low yield produced after the SEC process, we decided to carry out dialysis following IMAC, in an attempt to better produce protein for use in assays. The concentration of protein produced was:
Protein Concentration
Encapsulin 1.09mg/mL
LgBiT-Encapsulin 1.4mg/mL
SmBiT-Encapsulin 3.86mg/mL
Ultracentrifugation and SEC: The yields utilising IMAC were not great, which was attributed to the cage sequestering the His-Tag needed to associate with the nickel in the column. This was due to the His-tag being fused to the N-terminus which is internalised within the cage. In response to this another purification was performed using ultracentrifugation, with a gel run containing the purified samples after ultracentrifugation (see Figure 11), which led us to a discovery of the LgBiT protein not being present in all lab work leading up to this point (see LgBiT section below).
Figure 7: SDS-PAGE gel containing the products of Ultracentrifugation separated by their sedimentation fractions

The wild type encapsulin and SmBiT-encapsulin were further purified via SEC and then concentrated down:

Protein Concentration
Encapsulin 0.33mg/mL
SmBiT-Encapsulin 0.25mg/mL
Figure 8: SDS-PAGE of Encapsulin and SmBiT after SEC Red box indicates protein band

Although, this method produced protein of a greater purity, the yield proved to be weaker which meant the protein could not be used in assay testing or AUC.

Analytical Ultracentrifugation

While the LgBiT expression and purification required repetition, the SmBiT and wild type Encapsulin were pure and in relatively large concentration. This allowed us to perform analytical ultracentrifugation (AUC) to find out whether the purification was successful in producing functional encapsulin monomers (whether they form the cages the assay depends on) and whether fusing the SmBiT to the monomers has any impact on the cage formation.

The experiment was performed using wild type encapsulin at 1μM and 10μM (Figure 15), and SmBiT-encapsulin at 1μM and 10μM (Figure 16).

Figure 9: Analytical Ultracentrifugation Data for Wild-Type Encapsulin. The top graph depicts the molecular weight of the protein in AUC. The bottom graph depicts its sedimentation coefficients

The data produced from the AUC was very promising, with the sedimentation coefficients (~50S) clearly displaying the presence of large oligomers in both the wild type and SmBiT-fused encapsulin samples. Whilst the data could not present us with exact values (~2 x 106 Da), we can use this information to confirm that we produced functional monomers and that the fusion containing SmBiT did not hinder the cage formation, both essential results for our assay development.

Assay Testing

We attempted to observe a glow through the use of a plate reader when mixing our LgBiT and SmBiT fusion proteins, however due to the issues surrounding the expression of the LgBiT-encapsulin protein, these were unsuccessful. As the repeat expression of the LgBiT is completed we will look to observe and record a glow before we travel to the iGEM Jamboree.