iGEM 2024 — Team unc-chapelhill

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{% block title %}Notebook{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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# NOTEBOOK

April 24

Wednesday

Central accomplishments(s)

* Overview and Preliminary Plans for Peptide Production and Binding Affinity Testing

Wet Lab

Today’s goal was to establish a comprehensive plan for producing a peptide with binding affinity to perfluorooctanoic acid (PFOA) using E. coli as the expression system. We outlined the following steps: preparation of the cell line, plasmid production for peptide transfection, and the use of polyhistidine tags for purification. Following the purification using His-tag column chromatography, we will explore methods for His-tag removal and verify its effectiveness. Quantitative analysis using a DC protein assay will determine peptide concentration and impurity levels. We will then conduct binding affinity tests using ELISA, with control groups, including cheaper molecules like palmitic acid. Biolayer interferometry will be used to measure the dissociation constant (Kd) for the peptide-PFOA interaction. The protocol remains subject to optimization as the experiment progresses.

April 2024
2

April 26

Friday

Central accomplishments(s)

* Plan for His-Tag Removal and Transcytosis Experimentation

Wet Lab

Today, we presented our experimental plans to the Sode Lab, focusing on the removal of the His-tag from the purified peptide using TEV protease, with reference to the NEB protocol. We selected the E. coli strain BL21 for peptide expression. An additional experiment was planned to investigate the transcytosis of the engineered peptide, following procedures from relevant mouse studies. We also considered direct peptide synthesis, with a final decision pending based on time and cost assessments.

May 16

Thursday

Central accomplishments(s)

* Plan for Transformation of E. coli BL21

Wet Lab

Our focus was on the transformation of E. coli BL21 with the plasmid containing the target peptide sequence. We decided to follow the detailed transformation protocol from Edvotek to ensure efficient transfection. The wet lab will proceed with this phase upon final review of plasmid preparations. In response to a request from Dr. Sode, the wet lab team was restructured. The new core members of the lab were identified as Grant Holland, Zaid Syed, and Nishant Dengi, with Sam Carew serving as an alternate. Other members are invited to shadow during research activities but will not actively participate in the wet lab moving forward.

May 2024
4

June 25

Tuesday

Central accomplishments(s)

* Determining Amounts of HSA and PFOA for Control Group

Wet Lab

The primary task for today was determining the required amounts of human serum albumin (HSA) and PFOA for control group experiments, referencing an article on HSA-PFOA interaction from ACS Publications. We also reviewed relevant data on albumin pocket Kd values to inform subsequent testing phases.

June 2024
5

June 26

Wednesday

Central accomplishments(s)

* Control Experiment Protocol and Team Meeting Notes

Wet Lab

The meeting today focused on finalizing the protocol for control experiments involving PFOA immobilization on a microtiter plate, using a human albumin ELISA kit. We discussed using PBS/MgCl₂ as a buffer and considered various surface material properties to optimize the binding of PFAS and albumin. Following immobilization, we plan to block non-specific binding sites and proceed with chemiluminescence measurements to generate a Kd curve for the control group. Testing will involve a range of albumin concentrations, with constant concentrations of PFOA and anti-albumin. The control group experiments will begin once we receive the recombinant protein vectors.

June 2024
6

August 21

Sunday

Central accomplishments(s)

* E. coli Transformation and DNA Production

Wet Lab

We are officially beginning to work in the Sode Lab. Today's efforts focused on attempting to produce more DNA using DH5α bacteria; however, the attempt was unsuccessful. This will necessitate ordering more DNA soon. The E. coli transformation procedure was initiated but yielded no positive results. A detailed review of each step has been noted to troubleshoot this issue, as colonies failed to form.

August 2024
7

August 22

Monday

Central accomplishments(s)

* AI Test Culture and IPTG Test Culture

Wet Lab

Both the 106Fa4 and 84Fa4 samples did not colonize as expected. In response, we began smaller-scale test cultures, with 5 mL AI solutions and IPTG cultures for protein expression assays. The tests involved adjusting media volumes and concentrations, but the smaller-scale tests showed discrepancies in optical density (OD). The AI culture met the required OD (0.4), while the IPTG culture remained suboptimal at 0.04. This outcome suggests a need for increasing cell density in future experiments to improve reproducibility.

June 2024
8

August 23

Tuesday

Central accomplishments(s)

* AI and IPTG Test Culture Results and Cell Lysis

Wet Lab

Today's results showed that only the AI solution achieved the target OD, while IPTG still fell short. Post-culture, we centrifuged the samples and added BugBuster to initiate cell lysis, followed by urea treatment for the insoluble fraction. The process successfully separated soluble and insoluble proteins, which will be analyzed using serial dilutions. The samples are now stored at 7°C for overnight analysis.

August 2024
9

August 27

Tuesday

Central accomplishments(s)

* Protein Quantification Challenges

Wet Lab

Our glycerol-stored pre-culture samples appear to have degraded, leading to an unsuccessful protein quantification attempt. Despite this, the assay data yielded an R² value of 0.98, indicating strong linearity in the standard curve. The data were normalized, and further dilutions were prepared for the next round of testing. We standardized protein quantification results by calculating the lowest adjusted concentration for all samples.

August 2024
10

August 28

Wednesday

Central accomplishments(s)

* E. coli Transformation Setbacks

Wet Lab

The E. coli transformation procedure was repeated, but once again, no colonies developed. Agar plates were remade, and the issue is under review.

August 2024
11

September 5

Thursday

Central accomplishments(s)

* Continued E. coli Transformation Setbacks

Wet Lab

Another E. coli transformation was performed. Plates were stored in a 4°C fridge with parafilm applied to prevent contamination. Several questions have been raised about the differences between proteins and their mutations, which will be discussed with Josh later this week.

August 2024
12

September 10

Tuesday

Central accomplishments(s)

* Adjusted Preparation for E. coli Transformations

Wet Lab

A fresh LB medium batch was prepared, and cultures were sterilized via autoclave in preparation for the next round of experiments. We have slightly adjusted the E. coli transformation procedure based on previous results, increasing the heat shock time to 1 minute and switching from LB to SOC medium to improve transformation efficiency.

September 2024
13

September 13

Friday

Central accomplishments(s)

* Miscommunication about DNA Fragments vs. DNA Plasmids

Wet Lab

A miscommunication with the dry lab was identified; instead of plasmids, DNA fragments were being inserted into E. coli cells, leading to failures in bacterial growth. This issue explains why none of the bacterial colonies survived on the kanamycin plates, as the bacteria lacked the resistance gene. PCR testing is planned to confirm no contaminants are present.

September 2024
14

September 19

Thursday

Central accomplishments(s)

* Restriction Enzyme Digestion and Results

Wet Lab

The first restriction enzyme digestion was performed on five vials. Each vial contained 1 µL of DNA and was digested using NC01 and HinD3. A 1% agarose gel electrophoresis was run, but visualization via UV light suggests more consistent dye incorporation is necessary for improved results.

September 2024
15

September 24

Tuesday

Central accomplishments(s)

* Ligation Challenges and Plasmid Formation

Wet Lab

The DNA ligation of PET-30a vector with the gene inserts was performed successfully. However, when transforming the DH5α cells, no colonies developed. It appears NC01 restriction enzyme was mistakenly used instead of Nde1, hindering the plasmid formation.

September 2024
16

September 25

Wednesday

Central accomplishments(s)

* DNA Quantification and Process Review

Wet Lab

No colonies developed again, prompting a DNA quantification test. Results indicated abnormally low concentrations, possibly due to incomplete ethanol washing during membrane preparation. We will repeat the restriction enzyme digestion and ligation, this time ensuring all reagent steps are performed correctly.

September 2024
17

September 26

Thursday

Central accomplishments(s)

* Shift to Differential Scanning Fluorimetry

Wet Lab

A discussion with Sode lab team members highlighted that using perfluoroalkyl compounds for an ELISA/BLI assay is not feasible due to size constraints. A pilot study using DSF (Differential Scanning Fluorimetry) will be conducted once protein expression is complete. We are still investigating how the engineered albumin can bind and remove PFAS from contaminated environments. We are also considering potential applications of our protein in environmental biosensors or water treatments that remain promising. Further studies will explore these areas once the protein is purified.

September 2024
18

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{% block title %}Engineering{% endblock %}
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# ENGINEERING

#### Contents

* [Overview](#overview)
* Designs
  + [Design 1](#Des1)
  + [Design 2](#Des2)
  + [Design 3](#Des3)
  [Design 4](#Des4)
[Design 5](#Des5)

[Design 6](#Des6)

[Final Design (Design 7)](#Des7)

[Design 8](#Des8)

## Overview

  During the need-scoping process of our integrated human practices, we engaged with a variety of professionals and literature, to isolate 3 areas of focus we could develop a solution for

* PFAS prevention: developing alternate materials to use instead of PFAS
* PFAS contamination: Finding ways to limit PFAS exposure through filtration
* PFAS bioaccumulation: determining a means to remove PFAS from the system

  After gaining knowledge about the variety of ways exposure is currently occurring, the severe health effects that can evolve with exposure, and the non-existent healthcare options available for PFAS removal, we decided on focusing on the development of a solution of PFAS accumulating in the body.

 Throughout the process of our protein design, we engaged with the guiding engineering principles of iGEM Design, Build, Test, and Learn in the development of a solution.
 Our initial ideas for a solution were inspired by nature, specifically, IgA antibodies. IgA antibodies enjoy high specificity and binding strength associated with antibodies, but also have the ability to be secreted from the bloodstream, where they are made, to the mucosa, where they serve as an immune defense.

 We wanted to create a protein with the same abilities as an IgA antibody: something with high specificity and ability to be transcytosis from the blood to the mucosa. We first tried to create a humanized antibody with a novel Fab site for PFOA. From our initial literature review, we learned that secretory IgA (sIgA) is a dimer, joined by a J chain peptide. The J chain peptide not only provides a physical scaffold to link the two IgA monomers into a dimer, but is also essential in transcytosis from the bloodstream to the mucosa, which is mediated by the polymeric immunoglobulin receptor (pIgR). pIgR, which is mostly found on mucosa cells (1), recognizes the J chain of sIgA dimers, which then signals associated transcytosis events in the mucosa cell.

![](https://static.igem.wiki/teams/5192/engineering/designprocess.jpg)

## Designs

## Design 1: A new IgA Antibody

 Build: We attempted to build a novel Fab using OptMAVEn-2.0 (2).

 Test: When using OptMAVEn-2.0, we ran into an impassable error since we were trying to build the antibody off non-protein residues (the PFOA molecule)
  Learn: We learned from further literature review that antibodies don’t typically bind to small molecules. We were not going to be able to use a traditional Fab. From our literature review, we learned that albumin is a natural protein that has the ability to bind to PFOA with a respectable binding affinity in the micromolar range. We then sought to complex the pocket of Albumin that binds to PFOA to a Fc domain region of antibody, since the Fc region of the antibody is where the J chain attaches and how pIgR mediates transcytosis.

## Design 2: A PFOA pocket complexed to human Fc

  Build: We designed a protein sequence for a pseudo-antibody that had a N→C continuous pocket of albumin that was found to bind to PFOA. The pocket cutoff parameters was simply that the secondary structures which contained the PFOA binding residues (which were identified in the paper which did crystallized the Albumin-PFOA complex) At the C terminus of this pocket we appended the hinge and Fc domain from a human IgA antibody. To build this model, we created the sequence and then predicted its structure with Alpahfold 2.

  Test: Before we ordered the gene sequence, we discussed it with our scientific advisors, and realized it would be extremely difficult to express IgA proteins in E.coli due to E.coli’s lack of post-translational protein machinery, the protein’s large size which could stress E. coli, and abundance of disulfide bonds, which are very difficult to form in the reducing environment of the E.coli. cytoplasm.

  Learn: We did further literature review. One option we considered was trying to express the chimeric protein in a new, more complex, expression system like yeast– but this would require considerable upfront costs. Fortunately, we found a paper describing an attempt to harness the transcytosis ability of pIgR through the attachment of a small peptide that could be attached to the C-terminus of an IgG antibody. We contacted the authors of the paper and set up a meeting with him. He told us how based on the results, we could get the transcytosis effect we desired with the minimal peptide sequence his lab used, as compared to the Fc domain.

![](https://static.igem.wiki/teams/5192/engineering/design2.png)

Design 2. Green is the IgA Antibody Fc and hinge region. Pink represents the minimal pfoa-binding pocket.

## Design 3: A PFOA pocket with a PASR peptide

 Build: We removed the hinge and Fc domain from Design 2 and replaced it with the peptide QRNPKLKLIRRHPTLRIPPI. The original moniker for this sequence was QRN, but for our project we have called it PASR (Peptide thAt triggerS pIgR-mediated transcytosis). The PASR peptide has been shown to bind to human and mouse pIgR at comparable affinities in-vitro. The PASR sequence has been shown to bind to pIgR and facilitate transcytosis when complexed to c-terminus of IgG in mice. The peptide has been optimized to express well and not aggregate.

 Test: We used the linker sequence used in the original paper, GGGGS, to link the PFOA-binding pocket to the PASR peptide. We created the sequence and appended it to both the N and C termini of the pocket and then folded the sequences with Alpha Fold 2.

 Learn: Likely due to its small size and abundance of charged residues, alpahfold2 predicted the PASR to have no secondary structure. For this reason, for further designs we decided to append the PASR to the N-terminus, as we did not want it to destabilize the C-terminus alpha helix (which has some residues in the binding pocket.) The N-terminus is further away from the binding pocket than the C-terminus.

 However, when reviewing the literature once more before ordering, we learned of a critical flaw in our design. Albumin’s 4 pockets for PFOA were not equal binding strength; one pocket had significantly higher binding affinity than the other 3. The pocket our chimeric protein was built off of was the low affinity binder.

## Design 4: High affinity PFOA pocket with a PASR peptide

 Build: We isolated the correct, high-affinity pocket of Albumin, which luckily enough was continuous from N→C. We could modify our design by simply replacing the old pocket with the new pocket. Like the first design, we defined our pocket to be the minimal number of residues needed to not disturb any secondary structures which the binding amino acids were contained in.

 Test: We tested appending the PASR peptide to both the N and C terminus. By this time, Alpha Fold 3 had been released, so we used Alpha Fold 3 to fold designs with the PASR sequence appended to either the N or C terminus with the GGGGS linker.

 Learn: In this pocket, both the N-terminus and C-terminus were predicted to be unstable when the PASR peptide was appended. This would be problematic since the N and C termini were both very close to the binding pocket, and an intrinsically disordered region (IDR) near this could cause the binding pocket to be unavailable as the IDR flailed around.

![](https://static.igem.wiki/teams/5192/engineering/design4.jpeg)

Albumin crystallized with PFOA (PDB ID: 7AAI) colored red, with the FA4 pocket colored blue. PFOA molecules are shown as spheres. Other ligands were removed for visual clarity.

## Design 5: Expanded High-affinity PFOA pocket with a PASR peptide

 Build: We could either expand the N or C terminus to add more space for the PASR peptide to move around without blocking the binding pocket. We decided to expand the C terminus for multiple reasons:

1. The residues further along Albumin in the C terminus direction faced the solvent, while the residues further along Albumin the N terminus direction made even more intra-protein contacts. We reasoned that residues that natively face water would be more stable than residues that make abundant intra-protein contacts that would be absent in our minimal pocket structure.
2. Expanding residues further along Albumin in the N terminus direction could increase the chances of unwanted fatty acid binding, since in the Albumin crystal structure we built our protein off of, myristic acid was observed in the FA3 pocket; we did not want to add more residues of the FA3 pocket and increase the likelihood of off-target binding.

 Test: We tested multiple lengths of an expanded c-terminus based on the same principle of keeping secondary structures intact. We predicted stability of the proteins by predicting the structure and assessing them in Alpha Fold 3. We predicted the structure of both the binding pocket with the PASR and linker on the N and the newly expanded C terminus.

 Learn: The pocket was most stable when we expanded the pocket structure by adding 2 alpha helices on the c-terminus side from the real albumin. Importantly, these residues have not been shown to bind to any other ligands, and resulted in a PASR peptide that was ~110% more further away from the PFOA ligand (~11 Angstrom vs ~23 Angstroms.)

 However, an old issue came up in our new design: there were methionines, cysteines and a disulfide bond in the pocket, which as previously mentioned, can cause folding issues when E.coli is used as the expression system.

## Design 6: LigandMPNN Redesigned PFOA+PASR chimera protein

 Build: We wanted to see if LigandMPNN could redesign the PFOA pocket and increase its binding affinity and also optimize for protein stability by removing cysteine and methionine. We input Design 5 into LigandMPNN with the default parameters, but restricted cysteines and methionines from being in our designed peptide sequences.

 Test: Before we ordered the designs, we realized we would not have the wet lab resources needed to properly utilize LigandMPNN, which requires considerable wet-lab based optimization after an initial screening is done

 Learn: Although we could not use LigandMPNN to increase binding affinity, we could still use it to redesign the non-binding residues for stability by removing the cysteines and methonines, which as previously mentioned cause protein folding issues in E.coli expression systems. The proof of concept for this approach has already been tested and requires only a fraction of the wet lab testing, which was a workload suitable for our lab.

## Final Design (Design 7): LigandMPNN Partial Redesigned PFOA+PASR chimera protein

 Build: We put Design 5 through the LigandMPNN algorithm, but fixed all residues within 7 angstroms of the PFOA ligand (except two cysteines that formed a disulfide bridge). We once again restricted cysteine and methionine from being incorporated into the generated protein sequences and took the fixed residues as context to build the rest of the protein.

 Test: LigandMPNN is a stochastic algorithm, meaning it does not converge on just one solution, but rather generates many sequences that could work. We folded 120 of these designs with Alpha Fold 3 and selected the top 4 designs with the lowest RMSD, highest pTm and highest plDDT scores to order for wet lab testing.

 Final Design Scheme: Residues colored green represent amino acids that were kept fixed during the LigandMPNN procedure.

![](https://static.igem.wiki/teams/5192/engineering/designscheme.png)

![](https://static.igem.wiki/teams/5192/engineering/structassign.png)

Structural alignment of final designed FA4 pockets (multiple colors) on the Native Albumin FA4 Pocket (Grey). PFOA is shown as spheres.

## Design 8: ELISA Sandwich Assay to test PFAS Binding affinity

 Build: Our initial plan involved using a sandwich ELISA to assess the binding affinity between our engineered binder, containing the FA4 pocket and PASR sequence, and PFOA/PFOSA. PFOA/PFOSA would be immobilized onto the ELISA plate (blue box) via physical absorption. The binder, which included a his-tag (black) from the plasmid for detection, would then be introduced. We intended to detect the binding event using an anti-his-tag antibody (yellow), followed by an anti-mouse IgG (brown) conjugated to an enzyme (red) that would catalyze a fluorescent reaction upon binding with substrate (purple). This fluorescence would provide a quantitative measurement of the binder's ability to interact with PFOA/PFOSA.

 Test: We planned to perform the ELISA in a sandwich format, where PFOA/PFOSA would be immobilized on the plate, and the engineered binder, with its his-tag, would bind to these molecules. The detection mechanism would involve a secondary antibody system to amplify the signal, providing measurable fluorescence when successful binding occurred. A control group using unmodified albumin would establish a baseline for comparison.

 Learn: However, through advice from a mentor, we quickly realized that the ELISA assay was not suitable for our purpose. The main issue was the size of the PFOA/PFOS molecules, which are significantly smaller than typical antigens used in ELISAs. For example, insulin (a small protein) is around 6 kDa, while PFOA is roughly 200 Da. ELISA is more appropriate for larger proteins, and the size of perfluoroalkyl compounds like PFOA makes it unlikely they could be effectively immobilized for a sandwich assay. Additionally, Biolayer Interferometry (BLI) faced similar challenges due to the small size of the molecules and the expense and availability of BLI tips. Based on this insight, we abandoned the ELISA and BLI approaches.

Instead, we shifted our focus to Differential Scanning Fluorimetry (DSF), which allows us to assess the thermal stability of the binder in the presence of PFOA. In DSF, we monitor tryptophan fluorescence as the protein is heated, tracking the transition from folded to unfolded states. A higher melting temperature (Tm) indicates greater stability, meaning better binding to PFOA. Our goal is to see a high Tm for PFOA, showing strong interaction with the binder, and a lower Tm for myristic acid, serving as a control to ensure specificity.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

# EXPERIMENTS

#### Contents

* Safety Procedures
  + [DH5α E.Coli](#safe-ecoli)
  + [General Safety Procedures](#general)
  + [PPE](#ppe)
* Emergency Procedures
  + [Bacterial Culture Spills](#bac-spills)
  + [Injury or Exposure](#inj)
* [PFAS Disposal Protocol](#pfas)
* [Wet Lab Planning/Data Analysis](#wetlab)
  + [E. coli Transformation Procedure](#ecoli-transf)
  + [LB Medium Preparation](#lb-med)
  [Preculture Preparation](#pprep)
[IPTG Solution for Test Cultures](#IPTG)

[Protein Quantification Assay](#quant)

[Serial Dilutions](#serial)

[NanoDSF](#NanoDSF)

- [References](#refs)

Prior to working with the E.Coli Strain we engaged with thoroughly engaged with legislation both from our local Health governing bodies (Environment Health and Safety (EHS), Institutional Biosafety Committee (IBC)) as well governmental regulations for the correct safety and disposal of bacteria and PFAS agents

## Safety Procedures

## Safety Procedures for working with DH5α E.Coli

DH5α Hazard Information:

 The E. coli strain, DH5α, is often used for throughput amplification of recombinant plasmid DNA Safe handling of these strains requires adherence to guidelines set by the NIH and rules relating to standard microbiological practices.

Important Relevant Guidelines:

* Appendix G-II-A-1. Standard Microbiological Practices (BL1)
* Appendix K-III. Biosafety Level 1 (BL1) - Large Scale

 The attenuated strains typically used in molecular biology are classified as Risk Group 1 (RG1) agents, which do not cause disease in healthy adults and require Biosafety Level 1 containment. However, genetic modifications that enhance virulence factors or alter gene expression regulation may increase invasiveness and virulence, necessitating stricter containment measures and safety protocols. This risk assessment does not apply to clinical isolates, which are classified as Risk Group 2 organisms with higher virulence, requiring Biosafety Level 2 containment.

 While RG1 laboratory strains of E. coli pose minimal risk, certain individuals, such as the young, elderly, immunocompromised, or those taking large quantities of antacids, may be at higher risk of infection. Inflammation can occur if the organism enters the body through a wound or injection. Ingestion may cause diarrhea, and skin infections or conjunctivitis may result from exposure.

## General Safety procedure

When handling E. coli strains, use appropriate safety precautions and good microbiological techniques:

A. Laboratory access is restricted at the discretion of the lab PI

B. Do not store food in the laboratory.

C. No eating, drinking, or smoking in the lab.

D. Avoid mouth pipetting.

E. Ensure all lab personnel are properly trained.

G. All bacterial work should be performed in a BSL-1 CabinetH Transport hazardous materials in sealed, labeled containers with secondary containment.

I. Handle liquids carefully to avoid splashes and aerosols; centrifuge with sealed tubes and rotors.

J. Handle sharps cautiously to avoid injury. Discard needles immediately into a puncture-resistant container and decontaminate by autoclaving or incineration.

K. Decontamination procedures:

 i. Decontaminate liquid cultures with 10% bleach for at least 30 minutes.

 ii. Dispose of solid waste (Petri dishes, loops, tubes, gloves) in autoclaved biohazard bags.

 iii. Clean work surfaces with 70% ethanol or fresh 10% bleach after spills or at the end of the day.

 iv. Transport materials to be decontaminated outside the lab in leak-proof containers.

L. Wash hands after handling viable materials or animals, after removing gloves, and before leaving the lab.

## Personal Protective Equipment (PPE)

A. Keep a spare change of clothes in case of contamination.
B. Wear lab coats and gloves when working with bacterial cultures.
C. Use safety glasses if splashes or aerosols are anticipated.
D. Dispose of contaminated gloves and lab coats in biohazard containers.
E. Disinfect cloth lab coats by soaking in 10% bleach for 30 minutes.
F. Do not wear personal protective equipment outside the lab.

## Emergency Procedures

## Bacterial Culture Spills

1. Alert nearby personnel.
2. Leave the area for 15 minutes to allow aerosols to settle. Replace contaminated PPE.
3. Upon return, cover the spill with 10% bleach.
4. Let it sit for 30 minutes.
5. Absorb the spill with paper towels and discard them in biohazard bags.
6. Use a broom and dustpan to clean debris. Dispose of broken glass in a sharps container.
7. Wipe the area with 10% bleach.
8. Dispose of contaminated PPE in biohazard bags for autoclaving.

## Injury or Exposure

1. Cleanse wounds: Wash thoroughly with antiseptic soap and water for 15 minutes.
2. Control bleeding.
3. Ingestion: Rinse mouth with water, but do not swallow.

## PFAS Disposal Protocol

The EPA recommends that researches and manufactures using PFAS and PFAS-containing materials prioritize disposal and destruction methods to minimize the potential for PFAS to release into the environment. The following technologies, listed in no particular order, within the categories of storage, underground injection, landfilling, and thermal treatment:

These technologies options for disposal of PFAS waste that generally have a lower risk of environmental release compared to other options (however, we do not have a access to many of these sites):

* **Interim storage with controls:** For PFAS containing materials with high PFAS content, storage can serve as a temporary solution. However, materials that are generated continuously or have high volume with low PFAS content may be less appropriate for storage.
* **Underground Injection Control (UIC)–Permitted Class I non-hazardous industrial or hazardous waste injection wells:** Class I wells are designed to isolate liquid wastes deep underground, protecting drinking water sources. This technology may be a viable option for managing PFAS-containing fluids, though it is not available in our research areas.
* **Landfills–Permitted hazardous waste landfills:** For wastes with relatively high PFAS concentrations, EPA recommends using hazardous waste landfills. However, research since 2020 has shown that all landfills release more PFAS than previously understood. However, the hazardous waste landfills do have leachate controls that reduce environmental releases, which is particularly important for certain PFAS materials that degrade more easily in landfill conditions.
* **Thermal treatment–Permitted hazardous waste combustors:** Recent studies indicate that certain thermal treatment units, when operated under specific conditions, are more effective at destroying PFAS and limiting releases. These include certain hazardous waste combustors and granular activated carbon (GAC) reactivation units, though uncertainties remain, such as the potential formation of harmful byproducts at lower temperatures.

Due to availability and cost, disposal of PFAS containing waste will primarily be to hazardous waste landfills.

## Wet-Lab Planning and Data Analysis

The primary goal of this project is to design and engineer antibodies that can bind to PFAS (Per- and Polyfluoroalkyl Substances), based on a thorough review of the scientific literature on PFAS, albumin, and antibodies. These antibodies will be tested experimentally, and the resulting data will be analyzed and visualized to assess their efficacy.

To begin, we need to confirm the proof-of-concept by evaluating whether existing literature and expert opinions suggest that our project could realistically work as a treatment for humans. If human application is not feasible, we will explore other potential benefits of engineering this antibody. Collaboration with the human practices team is essential during this stage to ensure that all ethical and practical concerns are addressed.

Next, we must identify the most suitable cell line for producing the antibody, optimizing both the production yield and the ability to culture the cells in the lab. Once a cell line is selected, the cells will need to be transformed to express the desired antibody. At this stage, we may need to consult with other Principal Investigators (PIs) to assist with specific challenges, especially if we decide to work with eukaryotic cell lines. It is important to keep in mind that prokaryotic cells, while easier to work with, have limitations in post-translational modifications compared to eukaryotes, which are generally more difficult to culture.

After identifying the appropriate cell line and achieving antibody expression, the focus will shift to isolating and purifying the antibody. This will involve developing a strategy to isolate the antibody from the cells and determining how to test for concentration and impurities. A thorough review of the literature will guide the selection of appropriate methods for this process.

Once the antibody is isolated, we will test its binding efficiency. ELISA assays come to mind as a potential method, but it is crucial to examine the literature to confirm how other researchers have validated antibody-antigen binding interactions. After identifying the correct tests, we will collaborate with the fundraising team, graduate students, and Dr. Sode to order the necessary reagents. It is also essential to design the experiments with appropriate negative and positive controls, as a robust experimental setup is critical to obtaining reliable results.

Finally, under the supervision of graduate students, we will conduct experiments to test a specific hypothesis regarding the antibody's binding affinity to PFAS compared to albumin. We will apply the necessary statistical tests to support our hypothesis and demonstrate the antibody’s efficacy. The final step will be to visualize and present the data in a way that clearly communicates our findings to a diverse audience, ensuring that the results are accessible and comprehensible.

## E. coli Transformation Procedure

#### Materials:

* E. coli competent cells (DH5α)
* Plasmid DNA (0.5 µL)
* LB medium (250 µL)
* Agar plates
* SOC medium (for updated procedure)
* 42°C heat block
* Ice
* Centrifuge
* Pipettes and tips
* 37°C shaker incubator

#### Procedure:

1. Thaw E. coli competent cells on ice.
2. Add 0.5 µL of plasmid DNA (volume: 75 µL) to the competent cells and gently mix by tapping.
3. Incubate the tube on ice for 30 minutes.
4. Heat shock the cells by incubating the tube in a 42°C heat block for exactly 45 seconds (increase to 1 minute in modified protocol).
5. Immediately place the tube on ice for 5 minutes.
6. Add 250 µL of prewarmed LB (or SOC for modified protocol) medium to the cells.
7. Shake the tube at 37°C for 1 hour at 225 rpm.
8. Centrifuge at 5,000 x g for 1 minute at 4°C.
9. Remove the top 100 µL of solution and mix the remaining portion.
10. Plate 100 µL of the remaining solution onto an agar plate.
11. Invert the plates and incubate at 37°C overnight.

## LB Medium Preparation

#### Materials:

* Soy peptone (10 g)
* Yeast extract (5 g)
* Sodium chloride (NaCl) (10 g)
* Milli-Q water (to make 1 L)
* Beaker (1 L capacity)
* Stir bar
* Graduated cylinder (1 L capacity)
* Autoclave
* 1 L bottle

#### Procedure:

1. Add 800 mL of Milli-Q water to a 1 L beaker.
2. Add 10 g of soy peptone, 5 g of yeast extract, and 10 g of NaCl to the beaker.
3. Stir the solution using a stir bar until well mixed.
4. Transfer the solution into a 1 L graduated cylinder.
5. Add Milli-Q water to bring the total volume to 1 L.
6. Decant the solution into a 1 L bottle.
7. Sterilize the solution by autoclaving.

## Preculture Preparation

#### Materials:

* LB medium (3 mL)
* Kanamycin stock solution (50 mg/mL)
* E. coli colony
* 37°C shaker incubator

#### Procedure:

1. Add 3 mL of LB medium to a sterile tube.
2. Add kanamycin to a final concentration of 50 µg/mL.
3. Inoculate the LB medium with a single E. coli colony.
4. Incubate the tube at 37°C and shake at 250 rpm for 16-18 hours.

## IPTG Solution for Test Cultures

#### Materials:

* LB medium (4.94 mL)
* Kanamycin stock solution (50 mg/mL)
* MgSO₄ solution (50 µL)
* Preculture (0.05 mL)
* IPTG stock solution

#### Procedure:

1. Add 4.94 mL of LB medium to a sterile tube.
2. Add 5 µL of 50 mg/mL kanamycin solution.
3. Add 5 µL of MgSO₄ solution.
4. Add 0.05 mL of the prepared preculture.
5. Prepare 2 tests per preculture.

## Protein Quantification Assay

#### Materials:

* Protein samples (insoluble and soluble)
* 660 nm Protein Assay Reagent
* 96-well microplate
* Pipettes and tips

#### Procedure:

1. Add 10 µL of each protein sample to individual wells of a 96-well microplate.
2. Add 190 µL of 660 nm Protein Assay Reagent to each well.
3. Mix and incubate for the recommended time.
4. Measure absorbance using a microplate reader.
5. Calculate the R² value to assess the correlation.

## Serial Dilutions of Protein Samples

#### Materials:

* Protein samples (soluble and insoluble)
* PPB (1x solution)
* Pipettes and tips

#### Procedure:

1. For insoluble samples: dilute 2 µL of the sample in 98 µL of 1x PPB (50x dilution).
2. For soluble samples: dilute 2 µL of the sample in 48 µL of 1x PPB (25x dilution).

## NanoDSF

#### Materials:

* NanoDSF instrument (e.g., Prometheus NT.48)
* Capillaries for nanoDSF
* Protein samples (with known concentrations)
* Buffer solutions (e.g., phosphate-buffered saline, PBS)
* Study-specific ligands or binding partners (e.g., PFAS, albumin)
* 0.2 µm filters (to remove particulates)
* Microcentrifuge tubes
* Pipettes and sterile tips
* Ice
* Centrifuge
* NanoDSF-compatible software (for data analysis)

#### Procedure:

1. In a 96-well plate, prepare the following mixtures:

* Protein sample (final concentration varies, to be optimized based on tryptophan residues in the protein).
* Ligand at a final concentration of 1 mM.
* Dilute both protein and ligand to their desired final concentrations using 1x PBS buffer.

2. Incubate the mixture on a shaker for 10 minutes to ensure proper binding between protein and ligand.
3. Carefully transfer the incubated samples into capillaries compatible with the Prometheus NT.48 NanoTemper instrument.
4. Load the capillaries into the Prometheus NT.48 NanoTemper differential scanning fluorimeter.
5. Set the instrument to measure fluorescence emissions at 330 nm and 350 nm as the temperature increases from 20°C to 95°C.
6. Set the ramp rate to 1°C/min for the scan.
7. Use the PR.ThermControl software to calculate the melting temperature (Tm) based on the thermal unfolding curve obtained from the fluorescence ratio (F350nm/F330nm).

[DOI: 10.1021/acs.chemrestox.3c00011](https://pubs.acs.org/doi/10.1021/acs.chemrestox.3c00011)

## Protocol for nanoDSF

* In a 96-well plate, prepare the following mixtures:
* **Protein sample:** (final concentration varies, to be optimized based on tryptophan residues in the protein).
* **Ligand:** at a final concentration of 1 mM.
* Dilute both protein and ligand to their desired final concentrations using 1x PBS buffer.
* Incubate mixture on a shaker for 10 minutes to ensure proper binding between protein and ligand.
* Carefully transfer the incubated samples into capillaries compatible with the Prometheus NT.48 NanoTemper instrument.
* Load the capillaries into the Prometheus NT.48 NanoTemper differential scanning fluorimeter.
* Set the instrument to measure fluorescence emissions at 330 nm and 350 nm as the temperature increases from 20°C to 95°C.
* Set the ramp rate to 1°C/min for the scan.
* Use the PR.ThermControl software to calculate the melting temperature (Tm) based on the thermal unfolding curve obtained from the fluorescence ratio (F350nm/F330nm).

For additional details on Differential Scanning Fluorimetry (DSF) in assessing PFAS/albumin interactions, refer to this publication: [DOI: 10.1021/acs.chemrestox.3c00011](https://doi.org/10.1021/acs.chemrestox.3c00011). They employ a similar method for evaluating such interactions.

## REFERENCES

1. [US EPA O. Interim Guidance on Destroying and Disposing of Certain PFAS and PFAS-Containing Materials That Are Not Consumer Products. 2020 Dec 15 [accessed 2024 Sep 29].](https://www.epa.gov/pfas/interim-guidance-destroying-and-disposing-certain-pfas-and-pfas-containing-materials-are-not)

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# RESULTS

#### Contents

* [Overview](#overview)
* [Ligand Docking and Δ G estimation](#liganc)
* [Immunogenicity](#Immunogenicity)
* [Results and Analysis](#results)
* [References](#refs)

## Overview

  After the design of our functional binder + transcytosis sequence (process mentioned in Engineering page) and the synthesis of our plasmid (described in Experimental) we engaged in both computational and wet lab procedures for the assessment of various properties. Through these methods we can divulge information like if the engineered pockets are predicted to bind to PFOA the same way as albumin, estimate the Δ G of binding for use in the model we developed (described in Modeling) which can tell us vital details regarding the thermodynamics of our binder, Albumin (PFAS-binding competitor), and PFAS itself within a transcytosis model.

## Ligand Docking and Δ G estimation

 We used RosettaLigand (1) to generate fifty ligand-docked protein predicted structures for each engineered pocket, and performed this for both PFOA and Myristic Acid. We refolded the sequences in Alpha Fold 2 (2) since structures generated with Alpha Fold 3 (3) are not permitted for use in docking; Alpha fold 2 structures had very high structural similarity to Alpha Fold 3(RMSD > 1.0 Å) and were used in docking.

  We used the FA4 pocket with PFOA from 7AAI (4) as the comparison structure for docking runs with PFOA, and used the Albumin FA4 pocket with Myristic Acid from 1BJ5 (5) as the comparison structure for runs with docking runs with Myristic Acid. We chose myristic acid as the comparison ligand since myristic acid was found to be co-crystallized with PFOA in Albumin (4).

  After generating the ligand-docked structures, we used PRODIGY-Lig (6) to estimate the ΔG values of binding for the top 3 structures from each ground which had the lowest ligand RMSD. The structures with the lowest ligand RMSD represent the structures generated by RosettaLigand with the most similar binding pose to the comparison structure. We reasoned these structures would provide the best estimates for ΔG since they are closest to the established binding mode from x-ray crystallography experiments.

![](https://static.igem.wiki/teams/5192/results/ligand.png)

  At first glance, the data suggests that our designed pockets bind myristic acid more strongly than PFOA, as indicated by the lower (more negative) ΔG values for MYR. However, the error margins represented by the RMSE are substantial, suggesting that the observed differences in ΔG between the two ligands may not be statistically significant. Specifically, the signal-to-noise ratio of 0.87, calculated by dividing the effect size (the difference between the predicted ΔG values of the two ligands) by the RMSE, indicates that the differences are within the noise of the model. This leads to the conclusion that while our models appear to bind myristic acid slightly better, this difference may not be meaningful due to the high uncertainty in the model’s predictions. Thus, we can say more confidently from the PRODIGY-Lig predictions that our engineered pockets likely have similar ΔG of binding values for both PFOA and Myr, even if we cannot pinpoint the exact value. Additionally, from these ΔG predictions, all four pockets would be predicted to bind to PFOA and Myristic Acid at similar strengths—no significant differences were predicted across designs.

## Immunogenicity

We predicted the immunogenicity of our proteins using AntigenPRO (7) which predicts the likelihood of a protein being recognized as a foreign antigen by the human body.

![](https://static.igem.wiki/teams/5192/results/table1.png)

Interestingly, the native FA4 sequence had the highest probability of being an antigen despite the fact it is a native protein that circulates the blood. However, this might be attributed to the fact that a considerable portion of the FA4 pocket does not face the solvent and instead makes contacts with the rest of the Albumin protein. Additionally, since LigandMPNN is trained on the Protein Database, which is biased towards globular proteins (8), the algorithm likely excels at designing globular proteins which are less likely to trigger an immune response than proteins with exposed hydrophobic regions. Indeed, recognition of solvent accessible hydrophobic areas of proteins plays a key role in how antigens are recognized and presented in mammalian immunity (9).

However, we need to be cognizant of limitations of AntigenPRO; although it was trained on a balanced dataset of antigens and non-antigens, it was primarily trained on bacteria toxin data.

We were also interested in understanding the basis for design 119’s lower estimated probability of being an antigen since each of the designs have relatively high sequence similarity (70-80%, not including the linker or PASR sequence.) We identified 6 instances of a unique amino acid in design 119 compared to the other 3 designs:

* K137 - others are N
* E1 - others are S, A
* R23 - others are E
* T50 - others are I
* R72 - others are K, E
* H74 - others are E, D

The substitutions that are the least synonymous are T50I and R23. As expected the residues project towards the solvent, and none of these residues face the ligand-binding pocket.
![](https://static.igem.wiki/teams/5192/results/des119.png)

## Results and Analysis

Our main results from the model is that we can determine the steady state concentration of PFOA in the bloodstream with parameters. Using literature-determined values for the dissociation constant of PFOA and albumin, along with their initial concentrations, we developed a plot to look at such PFOA concentrations. We note that since we do not have an exact value for our binder, we instead use a ratio to characterize our binder, where the ratio is defined as the dissociation constant of the binder-PFOA complex divided by that of albumin-PFOA complex. This is equivalent to the scaler S defined earlier and for the following example, this ratio is set is 2.0 and the transcytosis rate is kept at 0.1 uM/time:

![](https://static.igem.wiki/teams/5192/results/fig2.png)

Figure 2. Example Steady State Plot with Average Values with Ratio = 2.0. Initial concentrations of PFOA and albumin are set to the literature-determined average values. Final PFOA concentration accounts for both free and bound PFOA.

Indeed, as the initial binder concentration increases, the final PFOA concentration decreases in a nonlinear trend. However, additional analysis can provide insight into how effective the binder needs to be relative to albumin in terms of binding affinity. To provide a more complete picture of this aspect, we show how the variation of the ratio between the dissociation constants of the two complexes affects the steady state concentration of PFOA as shown in figure 3:

![](https://static.igem.wiki/teams/5192/results/fig3.png)

Figure 3. Initial Binder Concentration and Dissociation Constant Ratio Effects on Final PFOA Concentration. Initial PFOA and albumin concentrations were set to 1.0 and 63.9 uM, respectively.

Initial binder concentrations of 100 uM along with having a binding efficiency of approximately two times that of albumin with PFOA yields an approximately two-fold decrease in the amount of PFOA in the bloodstream. We also note that even with an unfavorable ratio of dissociation constants (greater than 1), there is still a decrease in the final PFOA concentration. Thus, for these conditions, we believe that our engineered binder has potential to effectively remove PFOA from the bloodstream, even if it is marginally better at complexing than albumin.

Using the same parameters, we were also able to determine the steady state concentrations of the engineered protein in the bloodstream. Having high levels of the binder may cause toxic effects, so being able to estimate the remaining amount of binder provides insight into how safe our engineered protein is:

![](https://static.igem.wiki/teams/5192/results/fig4.png)

Figure 4. Initial Binder Concentration and Dissociation Constant Ratio Effects on Final Binder Concentration. Initial PFOA and albumin concentrations were set to 24.1 and 63.9 uM and the transcytosis rate was set to 0.1 um/time.

Indeed, we see practically none of the binder remains in the bloodstream. Furthermore, even with the large initial binder concentrations, there is less than 10% that readily remains in the bloodstream, suggesting that the binder will not have a major toxic effect due to being in the bloodstream. However, since we can not determine the duration that it is in the bloodstream, we are unable to assess if it stays at high concentration for long periods of time. Rather, we can only conclude that steady state in the long-term is likely safe in terms of binder concentration.

Finally, since an experimentally determined value for transcytosis rate was not determined and given the lack of time scale, we test four discreet magnitudes of transcytosis rate and look at the effects on steady state PFOA concentration:

![](https://static.igem.wiki/teams/5192/results/fig5.png)

Figure 5. Effects of Changing Transcytosis Rate on Final PFOA Concentrations. Initial PFOA and albumin concentrations were kept at 1 uM and 63.9 uM, respectively.

Indeed, increasing the transcytosis rate increases the efficiency of the binder as for similar initial binder concentrations and ratios, we see that final PFOA steady state concentrations are smaller as the k4 value increases by magnitudes of 10 (emphasis on color map scale). Thus, we conclude the engineered protein has potential for efficient removal of PFOA from the bloodstream and modification to increase the transcytosis rate can make the binder more efficient in removal.

## REFERENCES

1. [Davis IW, Baker D. RosettaLigand docking with full ligand and receptor flexibility. Journal of molecular biology. 2008;385(2):381–392. http://dx.doi.org/10.1016/j.jmb.2008.11.010. doi:10.1016/j.jmb.2008.11.010](http://dx.doi.org/10.1016/j.jmb.2008.11.010)
2. [Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Žídek A, Potapenko A, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021 [accessed 2024 Sep 30];596(7873):583–589. https://www.nature.com/articles/s41586-021-03819-2. doi:10.1038/s41586-021-03819-2](https://www.nature.com/articles/s41586-021-03819-2)
3. [Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, Ronneberger O, Willmore L, Ballard AJ, Bambrick J, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 2024 [accessed 2024 Sep 30];630(8016):493–500. https://www.nature.com/articles/s41586-024-07487-w. doi:10.1038/s41586-024-07487-w](https://www.nature.com/articles/s41586-024-07487-w)
4. [Maso L, Trande M, Liberi S, Moro G, Daems E, Linciano S, Sobott F, Covaceuszach S, Cassetta A, Fasolato S, et al. Unveiling the binding mode of perfluorooctanoic acid to human serum albumin. Protein science: a publication of the Protein Society. 2021;30(4):830–841. http://dx.doi.org/10.1002/pro.4036. doi:10.1002/pro.4036](http://dx.doi.org/10.1002/pro.4036)
5. [NCBI/CBB/Structure group. 1BJ5: Human serum albumin complexed with myristic acid. Nih.gov. [accessed 2024 Sep 30]. https://www.ncbi.nlm.nih.gov/Structure/pdb/1BJ5](https://www.ncbi.nlm.nih.gov/Structure/pdb/1BJ5)
6. [Vangone A, Schaarschmidt J, Koukos P, Geng C, Citro N, Trellet ME, Xue LC, Bonvin AMJJ. Large-scale prediction of binding affinity in protein–small ligand complexes: the PRODIGY-LIG web server. Bioinformatics (Oxford, England). 2019;35(9):1585–1587. http://dx.doi.org/10.1093/bioinformatics/bty816. doi:10.1093/bioinformatics/bty816](http://dx.doi.org/10.1093/bioinformatics/bty816)
7. [Magnan CN, Zeller M, Kayala MA, Vigil A, Randall A, Felgner PL, Baldi P. High-throughput prediction of protein antigenicity using protein microarray data. Bioinformatics (Oxford, England). 2010;26(23):2936–2943. http://dx.doi.org/10.1093/bioinformatics/btq551. doi:10.1093/bioinformatics/btq551](http://dx.doi.org/10.1093/bioinformatics/btq551)
8. [Kirchmair J, Markt P, Distinto S, Schuster D, Spitzer GM, Liedl KR, Langer T, Wolber G. The protein data bank (PDB), its related services and software tools as key components for in silico guided drug discovery. Journal of medicinal chemistry. 2008;51(22):7021–7040. http://dx.doi.org/10.1021/jm8005977. doi:10.1021/jm8005977](https://pubs.acs.org/doi/10.1021/jm8005977)
9. [Tsan M-F, Gao B. Heat shock proteins and immune system. Journal of leukocyte biology. 2009;85(6):905–910. http://dx.doi.org/10.1189/jlb.0109005. doi:10.1189/jlb.0109005](http://dx.doi.org/10.1189/jlb.0109005)

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