Results

Generation of PCR products for use in Golden Gate assembly

The f3-55nm plasmid created by Dr. George P. Smith was used as a template to generate both PCR products that would be used during Golden Gate assembly. Both primers necessary for this experiment were created by IDT and targeted different locations on the main plasmid. Primer 1 generated a fragment 1,476 base pairs long, while Primer 2 generated a fragment 2,997 base pairs long.

Both products were visualized using a 1.5 % agarose gel to separate the PCR sample from the primers and other impurities (Figure 1). Although smearing occurred during several attempts of this experiment, we found that both PCR samples were observed on the gel and migrated to the appropriate positions with respect to the DNA ladder. It became clear that our samples contained the amplified fragments from the f3-55nm plasmid and were ready for purification

Figure 1: Gel electrophoresis of negative control (second well), Primer 1 PCR product (fourth well), and Primer 2 PCR product (sixth well).

Purification of PCR products

Our initial attempts to purify the samples was done using a gel extraction kit to excise the DNA from the gel and then purify it through several wash cycles. Unfortunately, this did not yield favorable results (Table 1) for a Golden Gate assembly and required us to troubleshoot our purification method. We eventually changed our purification protocol to a DNA/PCR cleanup using a DNA/PCR cleanup kit (T1030), which demonstrated significantly better results (Table 2). Given the favorable concentration and ratio of both PCR products, we decided to use them in the Golden Gate assembly experiment to construct our plasmid.

Table 1: Nanoquant results of DNA fragment product generated from second set of primers (product from first set of primers was not observed in the gel).

Table 2: Nanoquant results of DNA fragment products generated from first and second set of primers.

Construction of the fdGPS2.1(RGD4C)-Amp plasmid

Ligation of DNA fragments using Golden Gate assembly

Construction of the fdGPS2.1(RGD4C)-Amp plasmid involved the combination of both PCR products generated in previous experiments as well as four other sequences that were created by IDT. The volumes of each sequence was determined using Coleman’s Lab protocol to make an equimolar solution (Table 3). The restriction enzyme BbsI-HF (R3539S) was introduced into the solution to generate cuts in each sequence, as well as T4 DNA ligase (Mo202L) to recombine the newly modified sequences.

Table 3: Data involving sequences used for Golden Gate assembly (protocol referenced from Coleman’s Lab). PCR 1 fragment is bolded due to it having the highest ng/kb value and is a reference for other volume calculations.

Bacterial Transformation of fdGPS2.1(RGD4C)-Amp plasmid product

Once we completed the Golden Gate assembly, we transformed the fdGPS2.1(RGD4C)-Amp plasmid using NEB 10-Beta competent E. coli cells to test the viability and function of it. What we found was the plasmid did not convey ampicillin resistance to the bacterial cells as they died when incubated overnight on LB agar plates containing ampicillin (Figure 2). We believe this could have been due to several factors including the Golden Gate assembly experiment not being successful, potential errors made during the bacterial transformation experiment, and/or potential issues with the design of the fdGPS2.1(RGD4C)-Amp plasmid.

Figure 2: Bacterial transformation of fdGPS2.1(RGD4C)-Amp plasmid using NEB 10-Beta competent E. coli cells. The experimental plates at the top of the image (β-1 on the left and β-2 on the right) contained our plasmid and did not demonstrate any growth. The control plates at the bottom of the image (β + on the left and β - on the right) did not contain our plasmid, demonstrating growth for the positive control which contained pUC19 while demonstrating no growth for the negative control which contained nuclease-free water.

Next steps regarding generation and analysis of phage particles

Optimization of protocols

Although we did not succeed in producing a functioning plasmid that would generate phage particles, we have plans for progressing towards that goal. Firstly, our dry lab team will investigate potential issues that may have been present when designing the fdGPS2.1(RGD4C)-Amp plasmid, and make modifications to how the fragments combine during Golden Gate assembly (Figure 3). This will be done in parallel with the wet lab team, which will investigate potential issues associated with how the experiments are run and optimize them to improve our chances of the plasmid assembling.

Figure 3: Schematic design of fdGPS2.1(RGD4C)-Amp plasmid (designed using Benchling)

Purification & Analysis of fdGPS2.1(RGD4C)-Amp phage particles

Once we have successfully assembled our plasmid and transformed it, our next step would be to isolate the phage particles that are generated from the bacteria cells. We would achieve this using a PEG/NaCl isolation protocol to purify the phage sample. After purification, we would focus on quantifying the titer of phage particles present by using a double layer agar overlay assay. After determining the viral titer, we would use Electron Microscopy to obtain a real time image of our phage particle’s morphology and size. This experiment would also be accompanied by an SDS-PAGE to analyze the binding protein, RGD-4C, and antigens, TSOL18 and EG95, attached to the phage’s coat proteins. This would allow us to observe whether the peptides are present on the phage surface and if the phage itself is ready for functional testing.

To test for the function of the fdGPS2.1(RGD4C)-Amp phage, we would utilize a dendritic cell line and observe whether the cell binds to and engulfs the phage particles upon interaction. To determine if the phage was phagocytosed, we would add a fluorescent label to it and track the presence of fluorescence within the dendritic cell after the interaction has occurred.