Protein Structure Comparisons

ESR1:

ESR1–ESR1:

ESR1+300:

ESR1+300–ESR1+300:

For our cloning, our team constructed two fragments of the ESR1 gene (Full-length and ESR1 300+ fragment). For prediction of how their binding performance might differ, our team ran a simulation of the protein structure and dimerization of the ESR1 using AlphaFold Server, and contrasted the Full-length and 300+ fragment models using the confidence parameters pLDDT, pTM, ipTM, and the PAE matrix.
Corresponding to general forecasting, the 300+ fragment (ipTM = 0.83, pTM = 0.84) outperformed the predetermined cut-off value of 0.8, which denotes high-quality prediction, while the Full-length model (ipTM = 0.55, pTM = 0.55) did not cross the 0.6 value representing likely a failed prediction. These results indicate not only that the 300+ fragment exhibits high overall precision, but also that it has better confidence about how accurately the relative positions of the subunits are being anticipated, on the whole leading towards a better prediction outcome.
At the level of residues, a large part of the 300+-fragment model is shown in deep blue, indicating high confidence (high pLDDT). On the other hand, the Full-length model shows distinctly orange areas at the periphery, indicating the presence of low pLDDT values and lower confidence in those areas, thus indicating a uncertain structure prediction. The corresponding PAE plots highlight this difference even more. For the 300+-fragment, the PAE matrix shows low values for the estimation error for relative position and relative orientation between pairs of residues within the structure being predicted, which means high confidence for relative configuration of the dimer. For the Full-length model, on the other hand, distinctly high values of the PAE (brighter colors) are seen for inter-chain regions, indicating more uncertainty for relative orientation of the two monomers.
The analysis shows that the ESR1 300+ fragment is capable of assuming a more stable and accurate configuration, specifically exhibiting enhanced efficacy for complex formation. The truncated fragment exhibits high-confidence predictions (pLDDT, pTM, ipTM), indicating that AlphaFold predicts this variant to form a reliable homodimer interface with a stable overall structure. As such, the 300+ fragment model exhibits a stable dimeric structure (as shown by the high ipTM), without the uncertainty introduced due to the redundant regions inherent in the Full-length sequence. The ESR1 300+ truncated form achieves a stable predicted dimeric structure and is likely to accomplish its dimerization effect more than the Full-length protein.

Best Storing Method

The best method to store yeast is lyophilization(freeze drying). It is already widely used in biotechnology and it allows yeast to be stored for years in room temperature. Overall, vacuum sealed bags are the best storage container, while refrigeration is the best storage temperature. The best medium is synthetic designed (SD) medium. The best protective agent is trehalose.

To ensure long-term viability and consistent assay performance, we evaluated the optimal methods for lyophilizing and storing engineered yeast sensors. Below is a comprehensive protocol and analysis for effective freeze-drying and preservation.

Steps for Lyophilization

1. Cultivation

• Medium: Use YPD for general growth or SD medium for genetically modified yeast (e.g., with auxotrophic markers).
• Conditions: Incubate at 30 °C with shaking (200–250 rpm) until late log phase (OD₆₀₀ ≈ 1.0–1.5).
• Goal: Maximize cell viability and plasmid retention before harvest.

2. Harvesting

• Method: Centrifuge at 4000 rpm for 10 minutes.
• Washing: Resuspend pellets in sterile PBS or distilled water; repeat wash 1–2 times.
• Optional: Add selection markers for plasmid maintenance.

3. Protective Agent Addition

Add protective agents to stabilize membranes and proteins during dehydration.

4. Pre-Freezing

• Method: Rapid freezing at −40 °C or in liquid nitrogen.
• Container: Use sterile glass vials or cryo-tubes.
• Note: Ensure uniform freezing to prevent ice crystal damage.

5. Lyophilization (Freeze-Drying)

• Equipment: Laboratory freeze dryer with vacuum control.
• Cycle:
  • Primary drying: −40 °C → −10 °C under vacuum (ice sublimation)
  • Secondary drying: Gradual warming to 20 °C to remove residual moisture
• Duration: Typically 24–72 hours depending on volume and equipment.

6. Sealing and Packaging

• Immediately after drying:
  • Seal vials under vacuum or nitrogen.
  • Add desiccant if using pouches or bags.

Storage Conditions and Survival Rates

Packaging Types and Features

Recommended Accessories

• Desiccants: Silica gel packets to prevent residual moisture.
• Nitrogen flushing equipment: Reduces oxidation and extends viability.
• Vacuum sealing machine: Ensures airtight packaging.

Culture Media for Freeze-Drying Yeast

1. YPD Medium (Yeast Extract Peptone Dextrose)

• Composition: Yeast extract, peptone, dextrose.
• Advantages: Rapid growth; ideal for S. cerevisiae and engineered strains.

2. MRS Medium

Suitable for co-culturing with lactic acid bacteria or probiotic applications.

3. Synthetic Media (e.g., SD Medium)

Used for genetically modified yeast or precise nutrient control in research applications.

Protective Agents

• Trehalose: Replaces water molecules, stabilizes membranes and proteins; ideal at 5–10%.
• Glycerol: Prevents ice crystal damage and osmotic shock; also aids cold storage (5–10%).
• Sucrose / Lactose: Sugar-based stabilizers protecting proteins and DNA.
• Skim Milk Powder: Protein-sugar matrix cushioning cells during drying.
• Polyvinylpyrrolidone (PVP): Synthetic polymer that stabilizes proteins and nucleic acids.
• Sorbitol / Mannitol: Sugar alcohols reducing oxidative stress and stabilizing membranes.

3D Model

With the use of our system, for future development, we envision three BPA detection kits built upon our yeast two-hybrid plasmid system to bring convenience and reassurance to our community.

Outdoor Kit – Cartridge Tube with Dropper (Most Realistic Near-Term Version)

This is the most immediately achievable prototype you might focus on: A small transparent cartridge tube (1.5–2 mL) contains a lyophilized yeast pellet (with protectants + nutrients).
A disposable dropper is provided to collect ~1 mL of field water, which is added into the tube to rehydrate the yeast. After ~3–4 hours (or perhaps 4–6 hours) at ambient temperature (ideally 25–30 °C), the tube is observed: blue coloration indicates BPA, clear or colorless means no detection. Optionally, you can include a reagent vial or small bleach capsule to neutralize the yeast after reading for safer disposal.
If field temperature is low (e.g. less than 20 °C) , reaction time may slow significantly. You might need to instruct users to keep tubes in a pocket or under sunlight to warm. The pellet must carry sufficient nutrients (salts, carbon source, buffer) for yeast metabolism after rehydration. Color readout by eye in outdoor lighting can be ambiguous: faint blue may be hard to distinguish. You may include a color standard or smartphone-based reading aid. This version is your primary prototype goal, but still needs optimization and validation.

Home Kit – Pregnancy Test–Style Stick (Proposed Future Version)

This is a conceptual home-use device rather than a validated product. It’s modeled after rapid diagnostic test sticks: A slim, disposable stick (with cap) contains a sealed reaction chamber with lyophilized yeast + colorimetric substrate (e.g. X-gal or similar) and embedded nutrients. The user removes the cap, drops 2–3 drops of water (or water + buffer) into the chamber, and recaps. Over ~4–6 hours at room temperature (or slightly warmed), the yeast rehydrates and, if BPA is present, catalyzes a color change (blue) visible in the result window; otherwise the window remains clear.
4–6 hours is long for a home diagnostic — users typically expect results faster. In practice, signals may require even more time, especially at low temperatures. Ensuring even mixing, avoiding yeast clumping, and creating controlled fluid flow are engineering challenges. Yeast are cells, not just chemicals. The built-in bleach mechanism must be engineered to reliably kill yeast without leaking prematurely.

School Lab Kit – Transparent 12-Well ELISA Plate (3×4 wells)

This is a proposed classroom demonstration kit. Each well contains a pre-loaded, lyophilized yeast pellet (with embedded protectants and minimal nutrients). The wells may host different reporter yeast strains. In use: A student pipettes ~1 mL of a water sample (possibly buffered or minimal media + sample) into each well. The yeast rehydrate and begin sensing BPA overnight (e.g. ~12–24 hours) under controlled temperature (ideally 25–30 °C) in an incubator or warm spot.After incubation, wells expressing lacZ + X-gal turn blue, whereas negative controls remain colorless or light. Students compare which reporter (e.g. different promoters or reporters) responded most strongly or fastest.
The pellet must include dry nutrients or minimal media components (e.g. yeast extract, salts) for yeast metabolism; pure water alone may not support growth or reporter activity. Room temperature without incubation may be too slow; you need a warm environment (≥ 25 °C) to get a detectable signal in a reasonable time. The blue signal may be weak or delayed under suboptimal conditions (less oxygen, lower metabolic rate). This is a visionary prototype, not yet fully validated in all classroom settings.

Design Breakdown & Comparison

All of our potential kit ideas all point to the same goal– convenient and accessible. Thus, we compared our system’s estimate values and settings to current testing methods through chart to demonstrate distinct the pros and cons.

Just like what you can see in the chart, our system compared to other resources is cheaper, the waiting time is shorter, and storage is easier than in most lab settings, providing a more accessible method to the general public.

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