iGEM 2022 — Team stockholm

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# Notebook

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* [Bacteria cells](#tag1)
  [Mammalian cells](#tag2)

## Bacteria cells

[Notebook - bacteria cells](https://static.igem.wiki/teams/4214/wiki/notebook/bacterial-lab-book-final-compressed.pdf)

## Mammalian cells

[Notebook - mammalian cells](https://static.igem.wiki/teams/4214/wiki/notebook/cell-culture-lab-book-final.pdf)

[Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
[Parts](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)

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# Engineering Success

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* [Introduction](#tag1)
  [Engineering Cycle - Phase I](#tag2)
  [Engineering Cycle - Phase II](#tag3)
  [Engineering Cycle - Phase III](#tag4)
  [Engineering Cycle - Phase IV](#tag5)

## Introduction

Our goal was to engineer a recombinant intrinsic factor (BiG-IF) that the autoantibodies against pernicious anaemia (PA) cannot bind to, while maintaining its vitamin B12 affinity through site-directed mutagenesis (SDM). Unlike other systems, a biological system is more complex and challenging to engineer and with such systems, often our understanding is limited and the pace is reduced. Thus, it is important to have a comprehensive process with a very tight-knit feedback loop. This was accomplished by executing the carefully curated DBTL engineering cycle, consisting of design, build, test and learn stages, while running several feedback loops in parallel.

![engineering cycle](https://static.igem.wiki/teams/4214/wiki/engineering/engineering-cycle.png)

*Figure.* *Engineering cycle representing the design, build, test and learn stages*

## Engineering Cycle - Phase I

### Design

After a thorough literature review, IF mutants were designed using the BLOSUM62 substitution matrix to preserve the wild type structure as much as possible.

 IF being a mammalian protein, a display vector, pDisplayTM (display vector) from Thermo Fisher was selected as the transfection vector.

 A production vector, pSB1C3, was also chosen to enable the production of the protein in a bacterial system and for downstream purification and isolation.

 Wet lab experiments were designed to clone and amplify the wild type plasmids.

### Build

The mutant structures were built using AlphaFold.

 The IF gene was cloned into the display and production vectors using restriction cloning.

 The SDM was initiated for the mutants in phases.

### Test

Of all the structures built, the improperly folded mutants were excluded and the rest were further subjected to in-silico modelling.

 To assess the strength of binding, docking studies were carried out using Autodock Vina and Mutabind2 for B12 and cubilin receptor respectively.

 Immunogenicity studies were performed to check for introduction of novel epitopes in the mutants.

 The sequencing results from cloning of the display vector, revealed that IF had indeed been inserted into the expression vector but had an unintended stop codon after the IF sequence but right before the PDGFR sequence.

### Learn

Mutants binding affinities to both B12 and cubilin receptor, which are essential for the internalization of the B12-IF complex, were evaluated along with the immunogenicity scores and the most promising candidates were selected to be tested in the wet lab.

 The stop codon in the sequencing results of all the clones and the wild type resulted from the improper amplification of the IF gene fragment prior to the insertion of the newer restriction sites for cloning. This had to be removed to ensure a successful display of the protein.

 The mutations were unsuccessful in the first attempt at SDM as the primer design did not align with the SDM kit we were using.

 In our attempt to produce the mammalian protein, we chose a bacterial system that is proficient at production but it, theoretically, lacked an ability to produce a fully folded protein that can be successfully isolated and purified. This prompted us to pick a new plasmid, and a new strategy to bring the protein out into the periplasm using a PelB leader sequence.

## Engineering Cycle - Phase II

### Design

A new set of primers were designed carefully to possess the necessary overlapping bases at and around the mutation site.

 A new primer pair was designed to remove the stop codon from the expression vector.

 Wet lab experiments were designed to clone, mutate and amplify the wild type and the mutant plasmids.

 Transfection experiments were designed for HEK 293 suspension culture.

### Build

The mutations were incorporated into the expression vector in iterative steps from single to triple mutations with varying levels of progress.

 The stop codon from all the mutants were removed from the plasmids using SDM and the plasmids were plasmid prepped to ensure adequate concentration needed for transfection.

 Recombinant production of IF from BL21 cells was attempted with 2 different concentrations of IPTG with limited success.

 The mutant vectors were transfected into HEK 293F, suspension cells.

### Test

The sequencing of the mutations revealed that the SDMs were successful with the desired substitutions.

 The transfection efficiency appeared to be too low with minimal GFP being expressed.

 The results on the Western blot (WB) revealed that the IF is expressed on the surface as well as in the cytoplasm, which was undesirable as we needed maximum IF to be displayed on the surface.

 The flow cytometry results proved to be inconclusive.

 The recombinant production of IF in bacteria did not yield any definite band in the size range expected, hence warranting a change in strategy.

### Learn

The WB results confirmed that there was a band in the size range that we were expecting, but without any controls nor double antibody binding, we could not reliably conclude the results.

## Engineering Cycle - Phase III

### Design

A Pet26b+ plasmid was chosen to clone the IF gene containing the His-tag into and lead it into the periplasm.

 A positive control of the pure IF protein for the SDS-Page and WB was ordered.

 The WB was planned to run with antibodies against the IF protein and the Myc tag which immediately superseded the IF insertion in the expression vector.

### Build

The display vector was transfected into HEK 293 adherent cells and fluorescent WB and fluorescence microscopy (FM) was performed.

 The protein was produced using the new production vector against the previous vector in three different inducer concentrations.

### Test

The WB revealed that there was indeed a production of the variant 20201 but failed to show any wild type production.

 The HEK 293 adherent cells failed to adhere firmly to cover slips thereby not yielding any FM imaging.

 There was a band at the size range in the bacterial production, but it was getting eluted along with the other proteins at too low a concentration of imidazole.

### Learn

Poly-D-Lysine was utilized to coat the wells and the coverslips and FM was reattempted.

 A new wild type protein was used to generate a band in the desired size range.

 The wash buffer was diluted to ensure it left the IF intact in the column.

## Engineering Cycle - Phase IV

### Design

The protein production was initiated with 0.5mM of inducer as only that concentration yielded a band.

 Transfection experiments were initiated again with the poly-D-lysine coating of wells.

### Build

The display vector was once again transfected into HEK 293 adherent cells that were firmly attached onto the coating and FM was performed.

 The production of IF was re-initiated with a wash buffer concentration of 0.5 mM and an elution buffer concentration of 15mM imidazole.

### Test

The FM results revealed that there was expression of IF on the surface of cells.

 The images were preserved and taken forward for image analyses.

 This time, the protein failed to bind to the resin.

### Learn

Needs a re-evaluation and optimization of the protocol.

[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
[Dry lab](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27dry-lab%27%29%20%7D%7D)

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# Experiments

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* [Introduction](#tag1)
  [Bacterial Culture](#tag2)
  [In silico experiments](#tag2_2)
  [Plasmid amplification](#tag2_3)
  [IF gBlock amplification](#tag2_4)
  [Restriction cloning](#tag2_5)
  [Site directed mutagenesis](#tag2_6)
  [Protein production](#tag2_7)
  [Mammalian Cell Culture](#tag3)
  [Cell culture](#tag3_1)
  [Transfection](#tag3_2)
  [Western blot](#tag3_3)
  [Fluorescence microscopy](#tag3_4)
  [Flow cytometry](#tag3_5)

## Introduction

[Bacterial Culture](#tag2)
[Mammalian Cell Culture](#tag3)

Our experiments and approaches are shown on the following page with detailed protocols. They are divided into Bacterial culture and Mammalian cell culture experiments. The Bacterial part contains our general protocols, in silico as well as wet-lab experiments. The Mammalian part has our experiments from mammalian cell culture and contains cell culturing, transfection, western blot, fluorescence microscopy, and flow cytometry experiments. Some abbreviations that you will meet in the text are wtIF(wild type intrinsic factor), pDisplay mammalian expression vector (the plasmid from ThermoFisher). Each one of our protocols is accessible by clicking the button mentioning the protocol’s title.

## Bacterial Culture

The following protocols contain all of our detailed work starting from PCR and cloning until our protein production step.

### In silico experiments

[In silico cloning](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/in-silico-cloning.pdf)
[Primer design](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/primer-design-guidelines-and-principles.pdf)
[SDM primer list](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/primers.pdf)

### Plasmid amplification

[Transformation](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/transformation-of-e-coli.pdf)
[Liquid cultures](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/culture-preparation-overnight-bacterial-culture.pdf)
[Miniprep](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/miniprepp-protocol-qiagen-kit.pdf)

### IF gBlock amplification

[PCR](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/platinum-superfi-ii-green-mastermix.pdf)
[Gel electrophoresis](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/gel-electrophoresis.pdf)

### Restriction cloning

[Restriction Digestion](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/restriction-digestion-protocol.pdf)
[Gel extraction](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/gel-extraction-and-purification.pdf)
[Ligation](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/ligation-protocol.pdf)

[Transformation](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/transformation-of-e-coli.pdf)
[Colony PCR](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/colony-pcr.pdf)
[Liquid cultures](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/culture-preparation-overnight-bacterial-culture.pdf)

[Miniprep](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/miniprepp-protocol-qiagen-kit.pdf)
[Sequencing](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/preparation-of-samples-for-sequencing.pdf)

### Site directed mutagenesis

[Site directed mutagenesis](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/site-directed-mutagenesis-protocol.pdf)
[DPN1 Digestion](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/digestion-of-template-dna.pdf)
[Transformation](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/transformation-of-e-coli.pdf)

[Miniprep](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/miniprepp-protocol-qiagen-kit.pdf)
[Sequencing](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/preparation-of-samples-for-sequencing.pdf)
[Midiprep](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/midiprep-protocol-from-promega.pdf)

### Protein Production

[Production & Purification](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/g-if-production-and-purification-using-his-pure-cobalt-resin-column.pdf)
[SDS-Page Protocol](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/sds-page-protocol-for-protein-purification.pdf)
[Western Blot Protocol](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/western-blot-protocol-for-protein-purification.pdf)

## Mammalian Cell Culture

The following protocols contain all of our detailed work starting from cell culturing and transfection until fluorescence microscopy.

### Cell culture

[Adherent cells](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/culturing-cells-adherent-cells.pdf)
[Suspension cells](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/culturing-cells-suspension-cells.pdf)

### Transfection

[Adherent cells](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/transfection-adherent-cells-1.pdf)
[Suspension cells](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/transfection-suspension-cells.pdf)

### Western blot

[Adherent cells](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/western-blot-adherent-cells.pdf)
[Suspension cells](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/western-blot-suspension-cells.pdf)

### Fluorescence microscopy

[Fluorescence microscopy](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/fluorescence-microscopy.pdf)

### Flow cytometry

[Flow cytometry](https://static.igem.wiki/teams/4214/wiki/experiments/experiments/flow-cytometry.pdf)

[Software](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27software%27%29%20%7D%7D)
[Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)

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# Results

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* [Results](#tag0)
  [Bacterial culture results](#tag1)
  [pDisplay mammalian expression vector](#tag1_1)
  [PCR amplification of wt-IF gblock fragment](#tag1_2)
  [wt-IF gene into the pDisplay mammalian expression vector](#tag1_3)
  [Site-Directed Mutagenesis](#tag1_4)
  [Protein production and purification of wt-IF](#tag1_5)
  [Mammalian cell culture results](#tag2)
  [Cell culturing](#tag2_1)
  [Western blot](#tag2_2)
  [Fluorescence microscopy](#tag2_3)
  [Flow cytometry](#tag2_4)
  [References](#references)

## 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

![pdisplay-mammalian-expression-vector](https://static.igem.wiki/teams/4214/wiki/results/pdisplay-mammalian-expression-vector2.png)

*Picture.* *pDisplayTM mammalian expression vector. The size of the vector is approximately 5 kb*

We purchased from ThermoFisher Scientific our mammalian expression vector- pDisplay™ [[1]](#references). 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

![wtif-gblock-in-agarose-gel](https://static.igem.wiki/teams/4214/wiki/results/wtif-gblock-in-agarose-gel.png)

*Picture.*  *The wtIF gene from gblock was amplified by using two different set of primers*

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

![The wtIF gene cloned into the pDisplay mammalian expression vector](https://static.igem.wiki/teams/4214/wiki/results/the-wtif-gene-cloned-into-the-pdisplay-mammalian-expression-vector.png)

*Picture.*  *Agarose gel of the wtIF gene cloned into the pDisplay mammalian expression vector. The size of the PCR amplified IF fragment is ~1.4kb*

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.

![wtIF insertion into the pDisplay mammalian expression vector sequencing confirmation](https://static.igem.wiki/teams/4214/wiki/results/wtif-insertion-into-the-pdisplay-mammalian-expression-vector-sequencing-confirmation.png)

*Figure.*  *wtIF insertion into the pDisplay mammalian expression vector sequencing confirmation. The forward and reverse primers used for sequencing aligned on the in silico created TF\_IF\_wt plasmid which is the wtIF gene that was inserted into the pDisplay mammalian expression vector*

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

![mutant with plasmid ](https://static.igem.wiki/teams/4214/wiki/results/mutant-with-plasmid-backboine.png)

*Figure.*  *Snapgene sequencing confirmation of 9 variants (From top left: 201, 20201, 101, 10101, 20101, 203, 20203, 301 and 402)*

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

![western blot result](https://static.igem.wiki/teams/4214/wiki/results/wb-bb12-3.png)

*Picture.* *Western blot image from the protein production of wtIF in the pSB1C3 plasmid with the lacI promotor after IPTG induction. wtIF is detected around 55-70 kDa*

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]](#references) (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

![HEK 293T cultured and observed under 100X magnification](https://static.igem.wiki/teams/4214/wiki/results/a-b.png)

*Picture.* *Microscopy pictures [A and B] of HEK 293T cultured and observed under 60X magnification*

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

![western blot result](https://static.igem.wiki/teams/4214/wiki/results/wb.png)

*Picture.* *Western blot image of our samples- wild type, mutant 20201 and Negative control loaded with two different time points (24, 48) and concentrations (15 µg and 25 µg)*

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]](#references). The anti-mouse green fluorescence (1:1000) is emitted from the secondary antibody used against our specific myc mouse primary antibody [[3]](#references). The red color in the ladder is because of the PageRuler™ Prestained Protein Ladder [[4]](#references). 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

![fluorescence microscopy](https://static.igem.wiki/teams/4214/wiki/results/fm.png)

*Picture.* *Fluorescence microscope pictures of HEK 293T cells [A to F]. Scale is marked at the bottom left of the image. A: Negative control in Bright field B: Negative control overlapped with Bright field and DAPI staining, C: Positive control in GFP channel, D: Positive control GFP superimposed with DAPI staining, E: Variant 20201 in RFP channel, F: Variant 20201 superimposed with IF and DAPI staining.
The images were analyzed using FIJI-J imaging software [[5]](#references).*

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

[ ]
References

1. ThermoFisher Scientific
   **pDisplay™ Mammalian Expression Vector**
   *ThermoFisher Scientific*
   [Read it](https://www.thermofisher.com/order/catalog/product/V66020)

   ---
2. Atlas Antibodies
   **Polyclonal Anti-GIF Antibody**
   *Atlas Antibodies*
   [Read it](https://www.atlasantibodies.com/products/antibodies/primary-antibodies/triple-a-polyclonals/gif-antibody-hpa039908/?q=&t= )

   ---
3. ThermoFisher Scientific
   **c-Myc Monoclonal Antibody (9E10)**
   *ThermoFisher Scientific*
   [Read it](https://www.thermofisher.com/antibody/product/c-Myc-Antibody-clone-9E10-Monoclonal/MA1-980?imageId=28579)

   ---
4. ThermoFisher Scientific
   **PageRuler™ Prestained Protein Ladder, 10 to 180 kDa**
   *ThermoFisher Scientific*
   [Read it](https://www.thermofisher.com/order/catalog/product/26616)

   ---
5. C. A. ImageJ Schneider, W. S. Rasband, and K. W. Eliceiri, K. W
   **NIH Image to ImageJ: 25 years of image analysis.**
   *Nature Methods, vol. 9, no. 7, pp. 671–675, 2012*
   [Read it](https://imagej.net/software/imagej/)

[Parts](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)
[Proof of concept](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D)

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