EXPERIMENTS

  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

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. They employ a similar method for evaluating such interactions.

REFERENCES