iGEM 2025 — Team hsinchutaichung-twn

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#

* [Notebook](#Introduction)

# Notebook

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#

* [Overview](#Engineering)
* [Design](#Design)
* [Build](#Build)
* [Test](#Test)
* [Learn](#Learn)
* [Limitations](#Limit)
* [Concentrations Iterations](Iteration)
* [Robustness](#Robust)

# Engineering Overview

Our goal was to build a low-cost, yeast-based detection system that reports BPA with a visible output suitable for homes, schools, and community testing.

We engineered a yeast two-hybrid (Y2H) BPA sensor where BPA-induced dimerization of ERα (ESR1) reconstitutes a split transcription factor (LexA-BD/B42-AD) and turns on *lacZ*. A BPA-biased design (ESR1-300+ truncation + five LBD mutations at aa 519–535) produced a same-day visible response: **901 Miller Units** at 3 h with 1 μM BPA vs 17.9 MU control (~50×), while E2 stayed at 15.6 MU (~control). A full-length ESR1 version worked after overnight growth (129 MU; ~13×) but was slower and less specific.

To ensure fair comparisons across tubes, color readouts were normalized by reaction time, assay volume, and cell density (OD₆₀₀) and reported as Miller Units; when the plate reader saturated (“over”), we measured a 10× dilution and corrected.

We followed the iGEM engineering cycle throughout our process: **Design → Build → Test → Learn**.

![](https://static.igem.wiki/teams/6016/wikinfo3/unnamed90.webp)

# Design

We evaluated two ESR1 designs within the same Y2H architecture:

* **(i)** A truncated ESR1-300+ carrying five ligand-binding-domain substitutions (aa 519–535; Y, G, N, T, L; codons TAC, GGC, AAC, ACC, CTC) to bias for BPA and improve folding in yeast.
* **(ii)** A full-length ESR1 carrying the same five substitutions as an architectural control.

Both designs used identical bait/prey vectors and the same LexA-operator→lacZ reporter:

* **Bait (pGilda):** LexA-BD::ESR1-300+ (5×LBD) via EcoRI/BamHI
* **Prey (pB42AD):** B42-AD::ESR1-300+ (5×LBD) via EcoRI/XhoI
* **Reporter:** LexA operators → minP → lacZ → CYC1t
* **Mutagenesis:** Segmented site-directed mutagenesis to install five LBD mutations; assemble fragments.

*\*See process tab in our Wet Lab for more details.*

# Build

The bait plasmid (*pGilda*) encodes the LexA DNA-binding domain fused to ESR1, and the prey plasmid (*pB42AD*) encodes the B42 activation domain fused to ESR1. To bias the sensor toward BPA and improve folding in yeast, we engineered an ESR1-300+ truncation and installed five substitutions in the ligand-binding domain (aa 519–535; Y, G, N, T, L with codons TAC, GGC, AAC, ACC, CTC) using segmented site-directed mutagenesis.

Inserts were cloned into *pGilda* with EcoRI/BamHI and *pB42AD* with EcoRI/XhoI, maintaining reading frames at the BD/AD junctions. The reporter is a LexA operator promoter driving *lacZ* with a yeast terminator. All junctions were confirmed in-frame; promoters and terminators were correctly oriented; and no internal EcoRI/BamHI/XhoI conflicts remained post-assembly, as verified by diagnostic PCR, Sanger sequencing, and sequence alignment.

# Test

Each assay was run under four conditions (25–30 °C):

* **Blank:** Water/buffer only (matrix control)
* **Control (−ligand):** Yeast + substrate, no BPA/E2 (leakiness)
* **BPA 1 μM**
* **E2 1 μM** (estradiol; related molecule)

The *lacZ* reporter produces a yellow product visible to the human eye. We timed each reaction, read OD₄₂₀, and converted to Miller Units (MU = 1000 × OD₄₂₀ / [time × volume × OD₆₀₀]).
If the reader saturated (“over”), we measured a 10× dilution and used that value.
The Blank remained clear, and the Control of Mutant-300 was low, confirming negligible background and leak.

### Two Versions of the Sensor

1. **A) Mutant-300 (ESR1-300+ with 5 LBD substitutions):** Designed for BPA bias and improved yeast folding.
2. **B) Full-length ESR1 (same 5 LBD substitutions):** Architectural control; larger protein, typically slower in yeast.

For the full-length ESR1 build, we used the same vectors and restriction sites with the same five LBD substitutions, generating *pGilda–LexA-BD::ESR1-Full (5×LBD)* and *pB42AD–B42-AD::ESR1-Full (5×LBD)* analogs.

Mutant-300 cultures reached OD₆₀₀ ≈ 1 by ~3 h (ready for same-day reading), whereas the full-length set typically required longer growth (overnight) before a clear readout.

### A) Mutant-300 (ESR1-300+ 5×LBD)

### Visual Results

| Condition | Visual Readout | Interpretation |
| --- | --- | --- |
| Blank (dH₂O) | Clear | No background from solvent/matrix |
| Control (−ligand) | Very faint | Low basal leakiness |
| BPA 1 μM | Strong yellow | Robust activation of *lacZ* via Y2H reconstitution |
| E2 1 μM | Light yellow | Weak activation; notably less than BPA |

![](https://static.igem.wiki/teams/6016/wikinfo3/5a1407c1-9d8e-4883-a00a-165d0d3f56b5.webp)

### Quantitative Results (Miller Units)

| Condition | β-gal Units | Fold vs Control |
| --- | --- | --- |
| Blank | 0.0 | – |
| Control (−ligand) | 17.9 | 1.0× |
| BPA 1 μM | 901 | 50.4× |
| E2 1 μM | 15.6 | 0.87× |

**3 h — Mutant-300:** BPA = 901 MU (~50× control 17.9); E2 = 15.6 MU (≈ control).

![Mutant-300 assay results](https://static.igem.wiki/teams/6016/wikinfo3/screenshot-2025-10-08-at-20-20-09.avif)

### B) Full-length ESR1 (5×LBD)

### Visual Results

| Condition | Visual Readout | Interpretation |
| --- | --- | --- |
| Blank (dH₂O) | Clear | No background from solvent/matrix |
| Control (−ligand) | Faint | Slight basal color after long incubation |
| BPA 1 μM | Strong yellow | Sustained activation of *lacZ*; consistent with BPA response |
| E2 1 μM | Moderate yellow | Detectable activation, still less than BPA |

![](https://static.igem.wiki/teams/6016/wikinfo3/img-6459.webp)

### Quantitative Results (Miller Units)

| Condition | β-gal Units | Fold vs Control |
| --- | --- | --- |
| Control (−ligand) | 9.67 | 1.0× |
| BPA 1 μM | 129.3 | 13.4× |
| E2 1 μM | 49.7 | 5.14× |

**Overnight — Full-length:** BPA = 129.3 MU (~13× control 9.67); E2 = 49.7 MU (~5× control).

![Full-length ESR1 results](https://static.igem.wiki/teams/6016/wikinfo3/screenshot-2025-10-08-at-20-20-32.avif)

### Conclusion

The ESR1-300+ 5×LBD design meets our pre-set criteria (BPA ≫ control, BPA ≥ E2) and enables a same-day response.
The full-length version is functional but slower and less specific, making Mutant-300 more suitable for field use.
This matches our tube results: a single bright BPA tube at 3 h for Mutant-300, and broader yellowing at 24 h for full-length.

# Learn

The ESR1-300+ (5×LBD) design delivers a same-day, BPA-biased response with low background (901 MU at 3 h vs 17.9 MU control ~50×), while E2 remained low (15.6 MU). The full-length version functions but needs longer growth and shows higher E2 activation (BPA 129 MU ~13×, E2 49.7 MU ~5×), reducing specificity.

* Mutant-300 is the preferred design for same-day detection.
* Standardize a 3-hour color assay at 25–30 °C with visual or smartphone thresholding for field use.

# Limitations & Next Iterations

Our target users (homes, schools, community groups) need a sensor that works outside the lab—without incubators or cold-chain storage. Lyophilization (freeze-drying) turns yeast into a stable, shippable, room-temperature pellet that can be rehydrated on site.

Demonstrating that our Mutant-300 sensor retains performance after drying and storage is essential for a “fieldable, same-day BPA check.” Robustness testing answers four key questions:

* **Shelf life & storage:** Does the dried kit still work after weeks–months at room temp or 4 °C?
* **Transport tolerance:** Do pellets survive temperature swings and humidity?
* **Usability:** After rehydration with real samples, do we still get accurate results?
* **Reproducibility:** Are results consistent across batches and time?

# Concentrations Iterations

To move beyond a single “worked at 1 µM” result, we hope to run a 3-hour dose–response with the Mutant-300 design to quantify sensitivity and dynamic range.

BPA will be tested across 0–10 µM (e.g., 0, 0.05, 0.1, 0.3, 1, 3, 10 µM) alongside our standard Blank and −ligand Control, holding temperature (25–30 °C), assay volume, and timing constant.

For each tube we’ll record start/stop times, read OD₄₂₀, correct any saturated reads with a 10× dilution, normalize by OD₆₀₀, and report Miller Units (mean ± SD, n≥3).

From these data we’ll extract the EC₅₀ (midpoint), LoD (mean blank + 3 SD), and fold-activation vs control to define a practical same-day threshold.

Success looks like a clear, by-eye call at ≤3 h with LoD ≤ 0.3 µM, EC₅₀ ≤ ~1 µM, and ≥10× fold at 1 µM, confirming that users can detect BPA without instruments while our quantitative metrics back the visual readout.

# Robustness Iteration: Lyophilized vs Fresh

### Goal

Prove the kit works from a dry pellet.

### Change

Lyophilize cells with **5–10% trehalose** (± skim milk, sorbitol).
Trehalose replaces water and vitrifies membranes/proteins, preventing damage during drying/rehydration; skim milk/sorbitol cushion and osmo-protect cells to preserve the 3-hour BPA readout after storage.

### Build

Prepare pellets, vacuum-seal, and store at both **4 °C** and **room temperature**.

### Test

Rehydrate in sample; measure MU after **3 hours**.
Repeat after **1, 4, and 8 weeks** of storage.

### Success Metric

Lyophilized signal ≥ 70% of fresh; baseline unchanged.

### Decision

If passed, **freeze-drying becomes the default kit format**.

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{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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#

* [Protein Structure Prediction](#Protein)
* [Best Storage Method](#Storage)
* [3D Model](#Abstract)
* [Design Breakdown & Comparison](#Introduction)
* [References](#Reference)

# Protein Structure Comparisons

### ESR1:

![](https://static.igem.wiki/teams/6016/wikinfo3/unnamed70.webp)

![](https://static.igem.wiki/teams/6016/wikinfo3/unnamed71.webp)
![](https://static.igem.wiki/teams/6016/wikinfo3/unnamed72.webp)

### ESR1–ESR1:

![](https://static.igem.wiki/teams/6016/wikinfo3/unnamed74.webp)

![](https://static.igem.wiki/teams/6016/wikinfo3/unnamed73.webp)
![](https://static.igem.wiki/teams/6016/wikinfo3/unnamed75.webp)

### ESR1+300:

![](https://static.igem.wiki/teams/6016/wikinfo3/unnamed78.webp)

![](https://static.igem.wiki/teams/6016/wikinfo3/unnamed77.webp)
![](https://static.igem.wiki/teams/6016/wikinfo3/unnamed76.webp)

### ESR1+300–ESR1+300:

![](https://static.igem.wiki/teams/6016/wikinfo3/unnamed74.webp)

![](https://static.igem.wiki/teams/6016/wikinfo3/unnamed81.webp)
![](https://static.igem.wiki/teams/6016/wikinfo3/unnamed80.webp)

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

| Temperature | Expected Shelf Life | Viability Retained |
| --- | --- | --- |
| Room temp (25 °C) | 6–12 months | ~70–90% |
| Refrigerated (4 °C) | 1–2 years | ~90–95% |
| Frozen (−20 °C) | 2+ years | >95% |
| Ultra-low (−80 °C) | 5+ years | ~100% |

### Packaging Types and Features

| Packaging Type | Features | Notes |
| --- | --- | --- |
| Aluminum foil pouch | Moisture-proof, light-blocking | Ideal for bulk or food-grade yeast |
| Glass vials (sealed) | Airtight, inert | Common for lab-scale or pharma use |
| Vacuum-sealed bags | Reduces oxygen exposure | Add desiccant for extra protection |
| Nitrogen-flushed containers | Prevents oxidation | Best for sensitive strains |

### 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.

![](https://static.igem.wiki/teams/6016/images/7fdcafaf-f53b-42de-99d5-c9e589315244.webp)

### 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.

![](https://static.igem.wiki/teams/6016/images/44ecfc4b-0f9e-401b-8216-798ad901357b.webp)

### 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.

![](https://static.igem.wiki/teams/6016/images/112beca6-c897-40a1-bc55-7dd7e896cb68.webp)

# 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.

| Detection Method | ELISA | LC-MS | GC-MS | Yeast Sensor | Our Yeast Y2H |
| --- | --- | --- | --- | --- | --- |
| Cost per Test | USD $575 / 80 samples | ~USD $100–350 / sample *Outsourced testing fee* | ~USD $150–300 / sample *Outsourced testing fee* | Starting from €370 / up to 40 samples | ~USD 2 / sample |
| Storage Conditions & Shelf Life (Reagents) | ELISA reagents 2–8°C, 12 months | X | X | –80 ºC or 2–8 ºC | 4 °C |
| Complexity & Detection Time | Research use only. Add sample and antibody to ELISA plate, followed by washing, enzyme addition, substrate, and stop solution. 3.5–4 h (Sandwich ELISA); 2–2.5 h (Competitive ELISA). | Extraction, purification, concentration (sample preparation) | Extraction, purification, concentration, derivatization (sample preparation) | Relatively simple. Mix the sample with yeast, incubate, and read absorbance.  ~18–48 h | Very easy. Just add a drop of sample — detection by the naked eye.  a few hours |
| Sample Types | Biological samples, dietary food, water | Water, solid samples (sediment, sludge, particulates), biological samples | Biological fluids, tissues, environmental samples, food | Water samples or aqueous extracts | Water samples or aqueous extracts |
| Environment | Laboratory | Outsourced professional lab | Outsourced professional lab | Laboratory | Anywhere — only requires a matching reading device |
| Detection Range | 3.12–200 ng/mL | 0.25–10.0 ng/mL | 50–1000 ng/mL | 0–100 ng/mL | — |

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.

# References

* VidePak (PP Woven Bags). (n.d.).
  *Aluminum Foil Composite Bags: The Future of Protective Packaging.*
  <https://www.pp-wovenbags.com/the-differences-and-benefits-of-aluminum-foil-composite-bags-vs-woven-bags/>
* Oluwatosin, S. O., Tai, S. L., & Fagan-Endres, M. A. (2022).
  Sucrose, maltodextrin and inulin efficacy as cryoprotectant, preservative and prebiotic – towards a freeze dried *Lactobacillus plantarum* topical probiotic.
  **Biotechnology Reports**, 33, e00696.
  <https://doi.org/10.1016/j.btre.2021.e00696>
* Köhler, S., Schmacht, M., Malchow, S., Wolff, L., & Senz, M. (2020).
  Preparation of freeze dried and vacuum dried yeast starter cultures: evaluation of relevant viability detection analyses.
  **BrewingScience**, 73(3/4).
  <https://doi.org/10.23763/BrSc20-05koehler>
* N’Guessan, F. K., Coulibaly, H. W., Alloue-Boraud, M. W. A., Cot, M., & Djè, K. M. (2015, July 20).
  Production of freeze-dried yeast culture for the brewing of traditional sorghum beer, tchapalo.
  **Food Science & Nutrition**, 4(1), 34–41.
  <https://doi.org/10.1002/fsn3.256>
* Chin, Y.-W., Lee, S., Yu, H. H., Yang, S. J., & Kim, T. (2021).
  Combinatorial Effects of Protective Agents on Survival Rate of the Yeast Starter, *Saccharomyces cerevisiae* 88-4, after Freeze-Drying.
  **Microorganisms**, 9(3), 613.
  <https://doi.org/10.3390/microorganisms9030613>
* Bistan, M., Podgorelec, M., Marinšek Logar, R., & Tišler, T. (2012).
  Yeast Estrogen Screen Assay as a Tool for Detecting Estrogenic Activity in Water Bodies.
  **Food Technology and Biotechnology**, 50(4), 427–433.
  <https://hrcak.srce.hr/en/file/139069>
* Xenometrix AG. (n.d.).
  XenoScreen XL YES – Yeast Estrogen Screen Test Kit for the Detection of Endocrine Disruptive Chemicals.
  [Link](https://www.xenometrix.ch/shop/XenoScreen-XL-YES-Yeast-Estrogen-Screen-Test-Kit-for-the-Detection-of-Endocrine-Disruptive-Chemicals)
* New Diagnostics. (n.d.).
  A-YBS.
  [Link](https://new-diagnostics.com/a-ybs)
* Owczarek, K., Waraksa, E., Kłodzińska, E., Zrobok, Y., Ozimek, M., Rachoń, D., Kudłak, B., Wasik, A., & Mazerska, Z.
  Validated GC-MS method for determination of bisphenol A and its five analogues in dietary and nutritional supplements.
  **Microchemical Journal**, 180, 2022, 107643.
  <https://doi.org/10.1016/j.microc.2022.107643>
* Phenomenex. (n.d.). GC & MS in Drug Testing & Toxicology.
  [Link](https://www.phenomenex.com/knowledge-center/gc-knowledge-center/gc-ms-drug-testing-toxicology)
* Sinha, A. (2025, September 18). Cost of GC-MS Analysis: In-House vs. Outsourced Testing. ResolveMass Laboratories.
  [Link](https://resolvemass.ca/cost-of-gc-ms-analysis/)
* De Nys, S., Putzeys, E., Vervliet, P., et al.
  A novel high sensitivity UPLC-MS/MS method for the evaluation of bisphenol A leaching from dental materials.
  **Sci Rep** 8, 6981 (2018).
  <https://doi.org/10.1038/s41598-018-24815-z>
* Agilent Technologies. (2015). Monitoring of Bisphenol A and Its Analogues in Environmental Matrices Using the Agilent 6550 Q-TOF LC/MS.
  [Link](https://lcms.cz/paper/11769)
* ResolveMass. (n.d.). How Much Does LCMS Analysis Service Cost in 2025? A Buyer’s Guide.
  [Link](https://resolvemass.ca/lcms-analysis-service/)
* FN Test. (n.d.). ELISA FAQs.
  [Link](https://www.fn-test.com/faqs/elisa-faqs/)
* BioCompare. (n.d.). Bisphenol A (BPA) ELISA Kit from AAA Biotech.
  [Link](https://www.biocompare.com/25138-Assay-Kit/23913785-Bisphenol-A-BPA-ELISA-Kit/)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}

## What Should this Page Contain?

---

* Clearly and objectively describe the results of your experiments and research.
* Use tables, graphs, and images to visualize your data.
* Discuss the implications of your findings in the context of your project goals.
* Highlight any trends, patterns, or significant observations.
* Include an analysis summary section to synthesize your findings and discuss their overall significance.
* Outline your future plans for the project, including potential applications, further research, and development.
* Explain what went wrong and what you learned from these experiences.
* Discuss potential improvements or alternative approaches.
* Emphasize the importance of scientific honesty and transparency.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/results)
* [2024 Marburg](https://2024.igem.wiki/marburg/results)
* [2024 EPFL](https://2024.igem.wiki/epfl/results)
* [2024 UToronto](https://2024.igem.wiki/utoronto/results)
* [2024 Marburg](https://2024.igem.wiki/marburg/results)
* [2024 Sydney-Australia](https://2024.igem.wiki/sydney-australia/results)
* [2024 Toulouse-INSA-UPS](https://2024.igem.wiki/toulouse-insa-ups/results)
* [2024 BNDS-China](https://2024.igem.wiki/bnds-china/results)
* [2024 Vilnius-Lithuania](https://2024.igem.wiki/vilnius-lithuania/results)
* [2024 XMU-China](https://2024.igem.wiki/xmu-china/results)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/results)

{% endblock %}