Results

Results

This page presents all the results from our wet lab experiments in two parts: Bacterial and Mammalian. The Bacterial part contains our PCR, cloning, mutagenesis, sequencing and protein production results. The other part contains our western blot, fluorescence microscopy and flow cytometry results of our mammalian cell culture experiments. Some abbreviations that you will meet in the text are: wtIF (wild type Human intrinsic factor), pDisplayTM mammalian expression vector (The display vector from ThermoFisher), wild type plasmid (TF_IF_wt) is the wtIF gene into the pDisplayTM mammalian expression vector, IF is the intrinsic factor protein and HEK is Human Embryonic Kidney Cells.

Bacterial culture results

pDisplay mammalian expression vector

We purchased from ThermoFisher Scientific our mammalian expression vector- pDisplay™ [1]. The pDisplay™ vector (refered to as the display vector) helps the proteins to be expressed in the surface of mammalian cells using a signal peptide that transports the protein to the membrane and also couples it with the transmembrane domain of a membrane protein - Platelet-Derived Growth Factor Receptor (PDGFR). Once displayed, the interactors of those proteins can be investigated using different assays. Some of the other pertinent features of pDisplayTM vector are T7 promoter, SV40 ori, HA tag, multiple cloning site, myc tag, along with Ampicilin and Neomycin resistance. We amplified the vector in TOP10 E. coli cells and performed gel electrophoresis to ensure that we isolated our plasmid (refer to the image above). We realised that apart from our vector represented as “band of interest” detected around 5 kb, there was another band at 10 kb, which disappeared once the plasmid was linearized proving that the latter was an unspecific aggregation of our desired vector. We proceeded with gel extraction of only our clear band of interest in order to clone our gene into the plasmid.

PCR amplification of wt-IF gblock fragment

We purchased the wtIF gene from IDT with our extra modifications containing the desired restriction sites. We included a His-tag for protein production and purification of our protein in bacteria. Therefore, the primers we used to amplify the wtIF gene removed the His-tag and the intrinsic signal peptide IF had. As it is pictured in the figure above, after PCR amplification the size of wtIF gene is around 1.4 kb meaning that we successfully amplified our gene. Our next step was to perform a restriction digestion with BglII (ThermoFisher) and SalI-HF (ThermoFisher) both in the wtIF gene and the pDisplayTM mammalian expression vector. After this step the two parts were ligated together using T4 DNA ligase (NEB).

wt-IF gene into the pDisplay mammalian expression vector

This is the colony PCR agarose gel electrophoresis image of the colonies screened for the wtIF fragment. The plasmids were extracted and investigated for the insert using the primers used to amplify the wtIF gene. The lone band in the H10 lane reflects the IF fragment in the solitary clone. This clone H10 contained the plasmid with the wtIF gene successfully cloned into it.

The H10 clone was sent for sequencing to ensure that our wtIF gene was inserted succesfully without any unintended mutations. The sequencing results using the forward and reverse primers yielded a good quality ~800 bp fragment that aligned with the in silico plasmid. This confirmed the successful cloning of the gene into the plasmid. Since we knew that our gene was present, we proceeded with site-directed mutagenesis to generate our mutant IFs.

Site-Directed Mutagenesis

These are the mutants that were carried forward for testing our binding assay. We tested the validity of those mutants by sending them for sequencing. In the picture above are shown the primers that we used to create them as well as the sequencing results highlighting the substitutions. The result shows that the necessary substitutions have taken their positions in the mutants. These mutants were amplified in TOP10 E. coli cells and forwarded to transfection in mammalian cells.

Protein production and purification of wt-IF

In order to implement our idea and have our protein into a pill, we also attempted to produce and purify the wtIF. The same procedure will be used to produce and purify the best mutant. For that reason, we used the pSB1C3 BioBrick plasmid with the LacI promotor. This promotor is inducible by IPTG, which will drive the protein expression, and the gene of interest. Protein production was initiated using this plasmid. As a negative control we used the pDisplay™ mammalian expression vector and the pSB1C3+LacI without the wtIF. A recombinant gastric intrinsic factor protein (from Sinobiological Cat: 13544-H08H) was used as a positive control for migration size and antibody binding. After the induction of the protein expression with IPTG, we purified our protein using HisPure Cobalt Resin. During the purification we collected the cell pellet, the flow through and the eluted part of the purification column. We loaded all of our samples in the SDS-page and followed it with a western blot. We used the epitope unspecific intrinsic factor rabbit primary antibody [2] (1:100) and anti-rabbit HRP(1:5000) as a secondary antibody. The wtIF expression was seen at the inducer concentration of 0.5 mM. The wtIF is detected around 55-70 kDa in the lysate and flow through of the 0.5 mM IPTG samples but not in the eluted parts of the purification.

Mammalian cell culture results

Cell culturing

The figures A and B successfully establish the good morphology of HEK 293T cells under 60X magnification. The figures A and B are taken at varied locations in different wells. These cultured cells were used for transfection and were further used for validation studies like fluorescent microscopy and western blot.

Western blot

Here, the anti-rabbit red fluorescence (1:1000) emitted is from the secondary antibody used against our epitope unspecific intrinsic factor(IF) rabbit primary antibody [2]. The anti-mouse green fluorescence (1:1000) is emitted from the secondary antibody used against our specific myc mouse primary antibody [3]. The red color in the ladder is because of the PageRuler™ Prestained Protein Ladder [4]. The prestained protein ladder is seen in the 700 nm range under fluorescence and hence they appear red in the blot.

Upper image depicts the western blot image (fluorescent image) of our samples- wild type plasmid, mutant 20201 plasmid and negative control (without any plasmid) loaded at two different concentrations-15 µg and 25 µg, and the control housekeeping gene GAPDH at 35 kDa. The samples were also collected at two different time points 24 and 48 hours (transfection time points). The ladder in kDa is marked along both the sides of the blot. Lower image depicts the same blot in a much enlarged view but without the control GAPDH gene.

From the western blot images, we observed that our protein (marked by green arrows) is being produced in mutant 20201 by the presence of light yellow bands in between 70 kDa to 55 kDa bands. The light yellow bands indicate the binding of both myc antibody (green) and epitope unspecific IF primary antibody (red). Myc is present in the plasmid backbone and is one of the tags for confirmation for our protein. However, the streak of green bands seen in the lower image can be understood as unspecified myc binding to various other cellular proteins in HEK cells. There is no red streak of bands seen in wells designated for samples thus indicating that our epitope unspecific antibody is extremely specific to the IF and does not bind to any other cellular proteins.

It is worthy to note that at the desired size range, we have no visible bands in negative control as well as in wild type plasmid in both figures. One of the reasons for no protein production in wtIF plasmid can be attributed to the stop codon removal step in our mutagenesis. While we succeeded in the removal, we believe that it may have caused some changes in the plasmid which we overlooked.

Fluorescence microscopy

From Figure A we can confirm that our cells are adherent and in good morphology. From Figure B, with the superimposition, we can establish that the blue DAPI (4′,6-diamidino-2-phenylindole) staining was successful as every cell appeared to be stained. In Figure C, we can see the bright green fluorescence courtesy of the Green Fluorescence Protein (GFP- the positive control used for transfection), confirming the success of our transfection. In Figure D, superimposing the GFP with blue DAPI was done to see the transfection efficiency visually. As one can observe, the transfection efficiency is not as high as expected for these cells. In Figure E, in the red fluorescence spectrum, we can see our IF protein. These IF proteins were tagged with primary rabbit anti-IF epitope unspecific antibody (1:100) and with secondary anti rabbit in red fluorescence (1:400). This confirms that our IF protein is expressed, thus coinciding with our western blot results as well. Figure F displays the superimposed image of DAPI staining the nuclei (blue) and the expression of IF on the surface of mutant 20201 (red).

Flow cytometry

We also performed the flow cytometry assay in HEK 293 suspension cells. However, the results weren’t conclusive. The positive control was expressed and gave some signal but neither the wild type plasmid nor the mutants were indicative of any expression.

References