iGEM 2023 — Team nyu-new-york

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{% block title %}Notebook{% endblock %}
{% block lead %}Hi all! Welcome to our Notebook Page. Below is an interactive google calendar where we've added events for all the big tasks we've worked on!{% endblock %}
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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Engineering design cycle seen through our project as a whole and for a specific part.{% endblock %}
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## Broad (Overall Project)

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### Define:

The issue we are addressing is that there are not enough cheap, at-home iron level testing kits that don’t involve drawing blood.

### Ask:

Approximately 25% of the world population is iron deficient (StatPearls) with women, children, and people in lower socioeconomic settings being more susceptible (and having less access to good healthcare on the global scale). This is a problem because many people might not have funds or insurance, blood drawing is dangerous for anemic people, and the current devices being hard-to-use makes them less accessible and reliable. There are also many countries where political and human rights issues prevent women, who are more likely to suffer from anemia, from having easy access to a doctor. This issue is compounded by the fact that people in lower socioeconomic classes are often the ones most affected by anemia and are the least likely to test their iron levels as it usually requires insurance or easy access to a free health clinic that tests these diagnostics.

### Imagine:

Our goal is to create a cheap, electrical, biosensor that detects one specific iron level marker found in saliva. We believe it is realistic to start off with one marker as we can develop the test to be more accurate and it will increase an individual's ability to understand their iron levels and make informed decisions. A more long-term goal would be to potentially detect more iron level markers depending on the data supporting our ability to do so.

### Plan:

Our plan is to:

* Find this target marker - **done** - it’s salivary ferritin!
* Gain understanding of different biosensor designs - **in progress**

+ There is so much to learn about biosensors! One of our captains (also wet lab lead) is taking a course right now on biosensors and she’s learning so much from it!

* Find aptamers capable of binding to our marker (ferritin) - **done**

+ After much literature review and computational docking (will go into detail below), we found two!!

* Survey the public to get a better idea of this project’s importance - **in progress**

+ We are trying our best to reach out to more women, people of color, and people from lower socioeconomic backgrounds as their perspective is especially important to making our biosensor accessible.

* Test the aptamers to see what (binding, detection, selectivity, etc) works best - **in progress**

+ We are currently doing direct biotin-streptavidin ELISA to test the aptamers against native ferritin samples.

* Develop biosensor design - **in progress**

+ We can confirm that our goal to make the biosensor cheaper is to make it arduino based.
+ We will potentially be doing a fluorescent signal for detection as the devices necessary to do this are inexpensive.

* CAD Modelling the biosensor - **not started**
* Building the biosensor and testing the aptamer’s stability within in - **not started**

### Prototype:

Our prototypes include the two aptamers (one peptide, one DNA) we designed through literature review and computational docking (more below on this). Our biosensor prototype is still a work in progress as we are still researching different designs and it is partially dependent on the stability of whichever aptamer we end up selecting after testing.

### Test:

* Before finalizing our aptamer designs and ordering them, we tested their binding affinity to salivary ferritin using computational docking. After confirming that their binding affinities were reasonably high, we are now testing the physical aptamers against native ferritin (liver specifically - has similar composition to salivary ferritin) through ELISA and DNA shift assay. More details on this are provided below and on our experiments and results pages!
* As our biosensor is still being researched, we are not yet at this prototype’s testing stage. Our tests will mainly consist of usability, how stable the aptamer is within our biosensor, and the accuracy of the data produced.

### Improve:

We will continue to improve our overall project’s design as we get more results from our aptamer testing, feedback from our surveying, and later on, results from our biosensor testing. We recognize that a project like this is definitely not finished in the first attempt and within a year. It is a strong cause that our team is passionate about, so we plan to continue it to completion and run through multiple iterations until the final product fits our goals.

## Specific (Peptide Aptamer Design and Testing)

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### Define:

The next step after identifying the iron level marker (ferritin) we wanted to detect was finding an aptamer capable of binding to it. An issue we ran into is that there aren’t many commercially available and there is not much literature/research on small proteins or oligos that can bind to ferritin.

### Ask:

We decided on a peptide or DNA aptamer for our project as we believe aptamers are generally quicker and more selective with binding. Generally, antibodies are used to detect markers like ferritin, but they are much slower and less accurate. We’ve included resources that we used to learn more about the benefits of aptamers over antibodies (ie. Bauer et al. and Novaptech) in the references section.

### Imagine:

We decided to turn to proteins and DNA sequences that already exist and are capable of binding to human ferritin. Afterall, the human body is full of them. From this, we could identify the specific residues responsible for binding and design a smaller aptamer.

### Plan/Sequence of Events:

**What’s been done:**

We started off with an extensive literature review to find proteins and DNA in nature that bind to ferritin. We found that H Kininogen and Human Transferrin Receptor 1 are two proteins in mammals that are capable of binding to ferritin. We originally looked into the binding residues in transferrin receptor 1, however there was not much literature or research already done on this chain’s binding affinity with ferritin. When looking into H kininogen, we found that there was a certain type known as high molecular weight kininogen (HKa) that is capable of binding to ferritin and has a lot of research backing this binding ability.

HKa is an anti-angiogenic protein, so when ferritin is in excess (correlates to high iron levels), it binds to HKa and prevents this anti-angiogenic activity. We found that there were already a few papers looking into finding a more specific ferritin binding region on HKa. Through different experiments testing how different peptide sequences on HKa inhibit HKa’s binding with ferritin when added to the mix, Coffman et al. were able to find that the 22aa sequence HGHGHGKHKNKGKKNGKHNGWK inhibited HKa-ferritin binding the most. In other words, this 22aa sequence has a very high binding affinity to ferritin which was then proven through other experiments detailed in that paper. We believed that this was a good amount of evidence to move onto the computational docking stage with this sequence.

We then spent June and July doing computational docking to test the peptide against ferritin. We specifically used Autodock Vina to accomplish this. Our first hurdle was that there were no pdb files available of the entire ferritin structure. Ferritin itself is just made up of 24 subunits of two repeating proteins (light chain and heavy chain ferritin). So, our solution to not having a full ferritin pdb was to test the peptide against just light chain ferritin and heavy chain ferritin and use those results as partial confirmation on the overall binding ability of our peptide. We then used Chemdraw and Pymol to draw our peptide aptamer, clean it (remove water, polarize, etc), and convert it to a pdb file. After multiple experiments of the peptide in different conformations with the light and heavy chains, we got some of the data shown below.

A goal for the future if we use computational docking again is to find if there is a way to use Autodock on our HPC. We are very lucky at NYU to have an HPC. We want to utilize it for tests like this as each Autodock experiment usually took a day to run and that slowed our progress down a lot. We found that the binding affinities we got from the peptide-ferritin tests were much lower than the ones found in the research papers. We believe that the most likely cause for this is that we were testing the peptide against one light chain or heavy chain ferritin instead of multiple chains.

Our plan was to use AlphaFold or a similar software to create a multimer with multiple light and heavy chains. However, we were having issues with accessing the programming necessary to do this and it was already reaching the end of July. We decided to move forward with ordering the peptide from Genscript based on the computational docking data we already had and our PI agreed with this decision.Below, the first image is the computational docking results from one of our first Autodock tests between the peptide and heavy chain ferritin. After noticing that the magnitude of the binding affinity was lower than expected, we energy minimized the peptide structure using another software and tested it again with heavy chain ferritin (results in second image below). Unfortunately, the images we collected of the tests run with light chain ferritin were not properly saved. The affinity (kcal/mol) of the mode 1 peptide conformation with light chain ferritin was -4.8 (so around the same as heavy chain). We will be running more autodock tests along with the ELISA to recollect this data.

![no_em_peptide_heavy_chain](https://static.igem.wiki/teams/4820/wiki/engineering-page-photos/no-em-peptide-heavy-chain.png)

Autodock Binding Affinity Results of Non-energy Minimized Peptide Aptamer with Heavy Chain Ferritin

![em_peptide_heavy_chain](https://static.igem.wiki/teams/4820/wiki/engineering-page-photos/em-peptide-heavy-chain.png)

Autodock Binding Affinity Results of Energy Minimized Peptide Aptamer with Heavy Chain Ferritin

**To be done:**

We are now testing our peptide aptamer physically in the lab. We ordered the peptide with one end biotinylated from Genscript. We are doing direct biotin-streptavidin ELISA to test the peptide’s binding ability with ferritin.

### Prototype:

After our computational docking tests, we determined that the peptide aptamer is viable enough to go onto the next stage - physical testing. We ordered the peptide, along with a DNA aptamer (more details on contribution page), from genscript to test its binding affinity with ferritin in the lab. We ordered the peptide to have the N-terminal biotinylated so that we could use it in direct biotin-streptavidin ELISA. The peptide came lyophilized and was found to be soluble in PBS, DPBS, tris-HCl, ultrapure water, and saline. Below is an image of an energy minimized conformation of the peptide on Chem3D.

![em_peptide_aptamer_3d](https://static.igem.wiki/teams/4820/wiki/engineering-page-photos/em-peptide-aptamer-3d.png)

Energy Minimized 3D Structure of Peptide Aptamer in Chem3D

### Test:

We are now doing direct biotin-streptavidin ELISA to test the binding affinity of the peptide aptamer with native ferritin. Our standards are recombinant ferritin from a Thermo Fisher ELISA kit. We are testing the peptide aptamer’s binding ability against a control antibody known to bind to ferritin that came with the kit. After getting a standard curve from the recombinant ferritin standards given in the first experiment, we will start using the native ferritin (purified ferritin from human liver) that we ordered as both standards and samples. More details on our protocol and any results can be found in the experiments and results pages. Here is a short summary of the plates we’ve done so far to give more context for the improvements we’re making.

**First experiment:**

* Plate 1:

+ recombinant ferritin standards with antibodies (both are from kit)
+ Native ferritin samples at decreasing concentrations with three different peptide dilutions – we’re finding the ideal peptide dilution while testing its binding ability
+ Blanks along the bottom of the plate

* Plate 2:

+ native ferritin standards with antibodies (antibodies from kit)
+ Native ferritin samples at decreasing concentrations with three different peptide dilutions (same concentrations as plate 1) - we’re finding the ideal peptide dilution while testing its binding ability
+ Blanks along the bottom of the plate

### Improve:

**Problems encountered:**

* The Thermo Fisher ferritin ELISA kit we ordered didn’t come with a specific blocking buffer - we assumed that the ferritin sample diluent contained BSA blocking buffer based on what minimal information we could find on the reagents. It must have been enough, however, as there was a lot of background noise in the first two plates we ran.
* The native ferritin and antibody did not bind in the standard rows of the second plate even though the kit said its antibody was capable of binding to purified native ferritin. Because of this, we won’t be able to use the native ferritin and antibody as a positive control.

**Solutions:**

* We have ordered Casein blocking buffer to properly block the plate moving forward.
* Potential positive control: Order more recombinant DNA and antibody as those two definitely bind together. This option is expensive, however.
* Potential negative control: Having blanks in the plate (which we have been doing until now)

+ This isn’t as helpful as the positive control but it can be used to mitigate background noise.

## References

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Bauer, M. J., Strom, M., Hammond, D. S., & Shigdar, S. (2019). Anything you can do, I can do better: Can aptamers replace antibodies in clinical diagnostic applications? Molecules, 24(23), 4377. https://doi.org/10.3390/molecules24234377

Coffman, L. G., Parsonage, D., D'Agostino, R., Jr, Torti, F. M., & Torti, S. V. (2009). Regulatory effects of ferritin on angiogenesis. Proceedings of the National Academy of Sciences of the United States of America, 106(2), 570–575. https://doi.org/10.1073/pnas.0812010106

Dhar, P., Samarasinghe, R. M., & Shigdar, S. (2020). Antibodies, nanobodies, or Aptamers—Which is best for deciphering the proteomes of Non-Model species? International Journal of Molecular Sciences, 21(7), 2485. https://doi.org/10.3390/ijms21072485

Di Rienzo, L., Milanetti, E., Testi, C., Montemiglio, L. C., Baiocco, P., Boffi, A., & Ruocco, G. (2020). A novel strategy for molecular interfaces optimization: The case of Ferritin-Transferrin receptor interaction. Computational and Structural Biotechnology Journal, 18, 2678–2686. https://doi.org/10.1016/j.csbj.2020.09.020

Eberhardt, J., Santos-Martins, D., Tillack, A.F., Forli, S. (2021). AutoDock Vina 1.2.0: New Docking Methods, Expanded Force Field, and Python Bindings. Journal of Chemical Information and Modeling.

Montemiglio, L. C., Testi, C., Ceci, P., Falvo, E., Pitea, M., Savino, C., Arcovito, A., Peruzzi, G., Baiocco, P., Mancia, F., Boffi, A., Georges, A. D., & Vallone, B. (2019). Cryo-EM structure of the human ferritin–transferrin receptor 1 complex. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-09098-w

NOVAPTECH. (n.d.). Aptamers vs antibodies I Key advantages of aptamers. https://novaptech.com/aptamers-vs-antibodies-advantages

Ponczek M. B. (2021). High Molecular Weight Kininogen: A Review of the Structural Literature. International journal of molecular sciences, 22(24), 13370. https://doi.org/10.3390/ijms222413370

The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.

Tesfay, L., Huhn, A. J., Hatcher, H., Torti, F. M., & Torti, S. V. (2012). Ferritin Blocks Inhibitory Effects of Two-Chain High Molecular Weight Kininogen (HKa) on Adhesion and Survival Signaling in Endothelial Cells. PLOS ONE, 7(7), e40030. https://doi.org/10.1371/journal.pone.0040030

Torti, S. V., & Torti, F. M. (1998). Human H-kininogen is a ferritin-binding protein. The Journal of biological chemistry, 273(22), 13630–13635. https://doi.org/10.1074/jbc.273.22.13630

Warner MJ, Kamran MT. Iron Deficiency Anemia. [Updated 2023 Aug 7]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK448065/

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{% block title %}Experiments{% endblock %}
{% block lead %}Below is a detailed review of our ELISA protocol for testing the binding between ferritin and our aptamer. Details on our computational docking protocols and tools have been added to the model page.{% endblock %}
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# **Menu:**

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* [ELISA](#ELISA)
* [Computational Docking](#CompDock)
* [DNA Shift Assay](#DNA)

# **ELISA:**

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**Experiment:** Create an Enzyme-Linked Immunosorbent Assay (ELISA) to measure concentration of ferritin using the peptide ligand or DNA aptamer that we found through literature review and computational docking. Find out if peptide ligand/DNA aptamer successfully binds to ferritin. Find out whether peptide or DNA binds more successfully to ferritin.

## **ELISA Protocol Overview:**

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1. Ferritin is added to 96 well plates at different concentrations.

2. The aptamer (DNA or peptide), tagged with biotin, is added so that it can bind to ferritin.

3. A small protein called Streptavidin conjugated with an HRP enzyme is added and the streptavidin binds to biotin.

4. A substrate is then added to react with the HRP enzyme and produce a chemiluminescent signal proportional to the concentration of ferritin in the well.

5. A plate reader then reads the absorbance of the signal produced by applying a 450nm wavelength, and the ferritin concentration is determined based on a standard curve of absorbance vs concentration generated from standard (known concentrations) ferritin samples.

![elisa-experiment-steps](https://static.igem.wiki/teams/4820/wiki/experiments-page-photos/elisa-experiment-steps.png)

Visual of ELISA Steps Listed Above Made Using Biorender

## **Trial 1:**

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* We ran two plates.
* Used commercially available ferritin ELISA kit for reagents and standards (to create standard curve).
* ELISA kit contained recombinant ferritin to use as standard and biotinylated rabbit anti-ferritin antibody
* We tested the peptide on our samples in the ELISA (not the standards).
* We used native ferritin for our samples because ferritin in human saliva is native and not recombinant.
* Plate 1

+ Standards: Recombinant ferritin, antibody added.
+ Samples: Native ferritin, peptide added.

* Plate 2:

+ Standards: Native ferritin, antibody added.
+ Samples: Native ferritin, peptide added.

## **Plate Map:**

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* Ferritin added at concentrations in ng/mL shown below.
* Blank = no ferritin added, control (should result in absorbance reading of 0).

![elisa-trial-1-plate-map](https://static.igem.wiki/teams/4820/wiki/experiments-page-photos/elisa-trial-1-plate-map.png)

Plate Map for Trial 1 (Stadard was recombinant ferritin + antibody for plate 1, native ferritin + antibody for plate 2)

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# **Computational Docking:**

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We did not add much here on the computational docking done prior to ELISA here as we go into detail on the tools and protocols used on the model page.

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# **DNA Shift Assay:**

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**Experiment:** Run a DNA shift assay experiment in order to determine if the DNA aptamer we found through literature review and computational docking successfully binds to ferritin. This experiment uses an agarose gel to visualize the migration pattern of DNA, ferritin, and DNA ferritin complexes. Smaller sized samples migrate further through the gel because they more easily fit through the pores of the gel. Larger sized samples do not migrate through the gel as far as smaller sized samples because they have a harder time moving through the pores of the gel. The samples in the gel are seen as bands under UV light.

## **DNA Shift Assay Protocol Overview:**

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1. Tris EDTA (TE) buffer is prepared.

2. 1.5% agarose solution in TE buffer is prepared, and ethidium bromide (binds to DNA and ferritin to allow for detection of bands in gel under UV light).

3. The agarose was poured into a gel casting tray in order for the gel to set.

4. Samples were prepared in microtubes.

a. 6 Samples: Ferritin, DNA aptamer, and four different concentrations of ferritin with DNA aptamer added to allow for DNA to bind to ferritin.

b. The goal was to see the difference in migration in the gel between these samples.

i. DNA should migrate the furthest since it is the smallest.

ii. If DNA successfully binds to ferritin, the complex should migrate through the gel more slowly compared to ferritin on its own so the complex will not migrate as far in the gel.

iii. For the ferritin-DNA complex samples, there might also be two bands visible due to excess DNA that did not bind to ferritin.

5. 1x non SDS loading dye was added to each sample to ensure that samples were successfully loaded into the wells of the gel and so that band migration could be visible when running the gel. SDS denatures proteins to allow for each band of the protein to be visible in the gel. We needed an intact native ferritin structure to allow for DNA binding so we did not use SDS.

6. TE buffer was poured into the chamber so that it covered the gel.

7. All six samples were loaded into the wells (six wells total loaded).

8. The electrodes of the electrophoresis chamber were attached to a power source, and the gel was run at 100 Volts for an hour.

9. The gel was then visualized under UV light.

![dna-steps](https://static.igem.wiki/teams/4820/wiki/experiments-page-photos/dna-steps.png)

Visual of DNA Shift Assay Steps Listed Above Made Using Biorender

![chamber-photo](https://static.igem.wiki/teams/4820/wiki/experiments-page-photos/chamber-photo.png)

Photo Taken of Gel Electrophoresis Chamber Set Up

## **Trial 1:**

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* We ran one gel.
* We prepared and loaded six samples at 30uL per well
* Lane 1: DNA (1ug)
* Lane 2: Native Ferritin (40ug)
* Lane 3: Native Ferritin (5ug) + DNA (20ug)
* Lane 4: Native Ferritin (10ug) + DNA (20ug)
* Lane 5: Native Ferritin (20ug) + DNA (20ug)
* Lane 6: Native Ferritin (30ug) + DNA (20ug)

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## **ELISA Trial 1 Results:**

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### **Plate 1 Results:**

![elisa-trial-1-plate-1-results](https://static.igem.wiki/teams/4820/wiki/results-page-photos/elisa-plate-1-results.png)

ELISA (ferritin + peptide aptamer) Trial 1 Plate 1 (Standard was recombinant ferritin + antibody)

### **Plate 2 Results:**

![elisa-trial-1-plate-2-results](https://static.igem.wiki/teams/4820/wiki/results-page-photos/elisa-plate-2-results.png)

ELISA (ferritin + peptide aptamer) Trial 1 Plate 2 (Standard was native ferritin + antibody)

### **Notes:**

* Inconclusive results.
* The commercial kit that was used did not come with a blocking buffer, and relied on a sample diluent with some blocking buffer on it. Blocking buffer is needed to prevent nonspecific binding of antibody/peptide, streptavidin-hrp, or substrate on the wells by blocking out empty spots on the wells that ferritin did not bind to. The sample diluent in the commercial kit was ineffective at blocking the plate, so there was a lot of background noise on the plate when absorbance was read.
* This was especially seen in the blank control wells that should have had an absorbance reading of 0, but instead had some high readings of 0.3-0.5.
* Most samples in plate 1 had high absorbance readings, while most samples in plate 2 had very low absorbance readings even though samples on both plates had the same native ferritin concentrations and the concentrations of peptide added were the same as well.
* In addition, it was found that the constructed standard curve of absorbance vs concentration was not linear.

### **Future ELISA Experiments:**

* Use PBS Casein blocking buffer.
* After each incubation, wash more thoroughly to prevent any background noise in absorbance readings.
* Load ferritin at concentrations more similar to average human salivary ferritin levels.
* Try different peptide incubation times to evaluate how quick peptide can bind to ferritin.
* Perform ELISA using DNA aptamer with biotin conjugate.

## **DNA Trial 1 Results:**

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### **Results Viewed in Regular Lighting:**

* Image of the gel before viewed under UV light.
* Bands are visible due to loading dye.
* 2 bands are visible in the native ferritin + DNA samples.

![DNA-trial-1-daylight-results](https://static.igem.wiki/teams/4820/wiki/results-page-photos/gel-day-1.png)

DNA Shift Assay Results Viewed in Regular Lighting

### **Results Viewed in UV Lighting:**

* Image of gel under UV light.
* The results of this experiment might be successful, but additional runs need to be done to confirm.
* Second band did not show under UV light for the ferritin/UV complexes → the second band is most likely DNA that did not bind to ferritin, and since there is less DNA that did not bind to ferritin it did not show up under the UV light.
* The lane with ferritin loaded by itself (40ug) did not show up on the gel → this might be due to the ethidium bromide not effectively binding to ferritin which prevents UV detection of ferritin.
* The lane with DNA by itself did show up on the gel and is represented by one band. Therefore, we know that the ethidium bromide did successfully bind to the DNA oligo which allowed for UV detection.
* The samples of ferritin/DNA complexes produced 1 band in the gel. Since ferritin by itself was not able to be detected and DNA was able to be detected it is likely that for lanes 3 through 6 (samples that contained both ferritin and DNA), DNA was able to successfully bind to ferritin and that is why it showed up on the gel.
* It is also possible the bands seen in lanes 3-6 are just DNA by itself and ferritin cannot be seen. However, lane 1 shows a pure DNA band that traveled further down the gel compared to the bands shown in lanes 3-6, so it is possible that these bands did not travel as far because DNA was bound to ferritin, and a ferritin DNA complex is bigger than DNA by itself.
* Furthermore, the second band shown in lanes 3-6 in the image taken before the gel was viewed under UV light could show that there some DNA was bound to ferritin, and some did not bind (possibly due to an excess concentration of DNA added compared to ferritin).

![DNA-trial-1-UV-results](https://static.igem.wiki/teams/4820/wiki/results-page-photos/gel-uv-1.png)

DNA Shift Assay Results Viewed in UV Lighting

### **Future DNA Shift Assay Experiments:**

* Load various concentrations of the DNA aptamer on its own to understand the migration pattern of this oligo.
* Load various concentrations of ferritin to understand migration pattern.
* Run a gel with a dye that allows for clear visualization of both ferritin and DNA aptamer.
* Run another gel with a dye known to bind to DNA only and not protein so that only ferritin with bound DNA can be visualized in the gel.
* Load ferritin + DNA samples at different concentrations.
* These future experiments will give us more data to confirm binding of the DNA aptamer to native ferritin.

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