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Team UCopenhagen 2024: Parts Collection of MilkClear

Introduction

Our parts collection is a comprehensive and innovative toolkit designed to address critical environmental and health challenges, specifically focusing on the detection and detoxification of harmful contaminants such as polycyclic aromatic hydrocarbons (PAHs), dioxins, polychlorinated biphenyls (PCBs), and bisphenol A (BPA) . Leveraging synthetic biology, our collection includes an array of receptor modules, reporter modules, and detoxification enzymes, enabling the development of highly specialized biosensor devices. These devices not only detect contaminants but also actively detoxify them, making our parts both practical and forward-thinking solutions to address contamination in breast milk.

The importance of developing a biosensor that not only detects but also actively detoxifies contaminants in breast milk cannot be overstated. While detection is a crucial first step in identifying harmful substances, it only highlights the problem without offering a direct solution. By integrating a detoxification system, we take this innovation a step further, enabling immediate action to neutralize and remove dangerous compounds such as bisphenol A (BPA), polycyclic aromatic hydrocarbons (PAHs), and dioxins. These contaminants are known for their endocrine-disrupting effects, which pose serious health risks to infants, who are particularly vulnerable during their developmental stages. Our dual-function device called SUPERMOM not only provides mothers with peace of mind by ensuring that harmful substances are identified but also guarantees a practical and efficient solution to detoxify the milk before it reaches the baby, ensuring that the nutritional and health benefits of breastfeeding are preserved. This combination of detection and detoxification offers a comprehensive approach to safeguarding infant health in an increasingly contaminated world, setting a new standard in maternal and infant care.

Our contribution to the iGEM community

We have designed and submitted a collection of modular, well-characterized parts to the iGEM community, significantly expanding the toolkit for environmental sensing and detoxification. These parts are fully compatible with existing iGEM standards, enabling teams to incorporate them into a wide range of projects. Below are the key ways we contribute to the iGEM community:

1. Biosensor Modules : Our parts include receptor modules, such as pRET2-LexA-ERα(LBD), which allow teams to detect endocrine-disrupting chemicals like BPA. These receptor modules are engineered to bind to specific contaminants and initiate a measurable reporter response, using constructs like Lex6Op-pLEU2-NanoLuc to output luminescence when contaminants are present. This system provides a ready-to-use platform for teams aiming to detect environmental pollutants.

2. Detoxification Systems: In addition to detection, we offer detoxification modules, such as CYP1A1-pGAL1/10-POR and UGT2B15, which metabolize and neutralize harmful compounds like BPA, PAHs, and dioxins. These parts are especially useful for projects aiming to create solutions that go beyond detection to actively remove toxins from the environment.

3. Modularity and Flexibility: All parts in our collection are designed with modularity in mind, enabling teams to mix and match receptor modules, reporter modules, and detoxification enzymes to create customized biosensor systems. This flexibility makes our collection an essential resource for any iGEM team working on environmental sensing, bioremediation, or public health projects.

4. Cross-Project Usability: Our parts collection is adaptable to various chassis and biological systems, from Saccharomyces cerevisiae to other eukaryotic and prokaryotic hosts. This cross-compatibility ensures that iGEM teams from different domains can easily integrate our parts into their work, providing a universal toolset for detecting and managing environmental contaminants.

Showcasing the Impact of our Parts Collection

By contributing with this collection of receptor, reporter, and detoxification modules, we provide the iGEM community with tools that not only enhance current biosensing capabilities but also empower future teams to build more advanced, multifunctional systems. The potential applications of our parts extend from environmental monitoring to public health, making this collection a significant step forward in the fight against pollution and harmful chemicals. Our work encourages the iGEM community to explore synthetic biology as a practical solution for real-world environmental challenges, ensuring the well-being of future generations.

Devices

The Devices Table outlines the different biosensor devices developed for the detection and detoxification of various environmental contaminants. Each device incorporates specific receptor modules and reporter modules, tailored to detect particular compounds like polycyclic aromatic hydrocarbons (PAHs), dioxins, polychlorinated biphenyls (PCBs), and bisphenol A (BPA). For instance, devices BBa_K5477041 and BBa_K5477042 are biosensors designed to detect PAHs, dioxins, or dioxin-like PCBs, while devices BBa_K5477043 to BBa_K5477045 are specialized for BPA detection. Additionally, BBa_K5477046 introduces the SUPERMOM device, which integrates both detection and detoxification modules to simultaneously identify and neutralize BPA in breast milk. The final device, BBa_K5477047, is a detoxification device targeting PAHs, dioxins, and PCBs through the use of CYP1A1 and UGT enzymes, which metabolize these contaminants into safer, water-soluble compounds. This array of devices demonstrates the versatility of the biosensor platform in addressing diverse environmental and health-related issues.

Results

Findings on the pRET2-ERα biosensor

The pRET2-ERα biosensor demonstrates detectable responses to several environmental contaminants, including BPA, Aroclor 1260, and PAH. However, its performance is notably limited in comparison to alternative systems such as mERα and constructs using the pPOP6 promoter. The pRET2-ERα system shows sensitivity to BPA and Aroclor 1260, but its performance is reduced in complex environments like milk and plateaus at higher concentrations for PAH.

1. Response to BPA: The pRET2-ERα biosensor showed peak sensitivity at ~1 µM, with a dynamic range of 1700-7400 luminescence units, though no EC50 could be determined due to the absence of a plateau.

2. Response to BPA in Milk: In milk, pRET2-ERα exhibited reduced luminescence (200-300 RLU), but still showed an upward trend with increasing BPA concentrations, indicating diminished yet detectable sensitivity.

3. Comparison with mERα for BPA Detection: The mERα biosensor showed greater specificity and stronger signals for BPA detection compared to pRET2-ERα, especially in milk, where mERα was less affected by estrogen.

4. Promoter Optimization (pRET2 vs. pPOP6 for BPA and Aroclor 1260 Detection): The pPOP6-ERα produced higher signals than pRET2-ERα for both BPA and Aroclor 1260, with a gradual increase in signal for Aroclor, suggesting pPOP6 is more effective.

5. Response to PAH: The pRET2-ERα biosensor plateaued at higher PAH concentrations, indicating a need to test below 2.5 nM for better sensitivity assessment.

Findings on the pPOP6-ERα biosensor

The pPOP6-ERα system, on the other hand, exhibits stronger signals and greater sensitivity across a broader range of concentrations for both BPA and Aroclor 1260, indicating that it may be a more optimal choice for future biosensor development. The use of mERα over ERα is also recommended for BPA detection, particularly in environments where background estrogen may interfere with detection.

Findings on the pRET2-mERα biosensor

The engineered mERα biosensor demonstrates significant potential for detecting a range of environmental contaminants, including BPA, PCB3, PAH (benzanthracene), and Aroclor 1260. Its performance varies depending on the specific compound and the environmental matrix, such as the presence of milk. Across the experiments, several key findings were observed for each contaminant below. For more details, visit documentation for this device BBa_K5477045:

1. Response to BPA: The mERα biosensor exhibited a stronger response to BPA compared to the ERα biosensor, demonstrating higher specificity and selectivity towards BPA. A visual estimation suggested an EC50 of around 6500 luminescence units, though further validation is required to confirm this. The mERα system was able to produce a clear response to BPA, but showed a plateau in luminescence beyond 10 nM.

2. Response to BPA in Milk: The mERα biosensor showed a significant decline in luminescence as the proportion of milk in the assay increased, particularly at higher milk fractions. The biosensor maintained functionality in diluted milk, although the signal declined sharply at certain concentrations (e.g., 1/64 milk dilution). This suggests that the milk matrix may interfere with detection by either affecting yeast viability or luminescence measurement.

3. Comparison with ERα Biosensor: mERα was more selective to BPA than ERα and showed reduced activation by estradiol, supporting its potential for use in complex environments like milk, where background estrogen could be an issue. Despite a higher response to estradiol compared to ERα, mERα's strong response to BPA still suggests low background interference in milk.

4. Promoter Optimization: When comparing different promoters, the pPOP6-ERα biosensor showed higher potency in detecting BPA compared to pRET2-mERα. The findings suggest that mERα could potentially perform better if expressed under the pPOP6 promoter, indicating an opportunity for further optimization.

5. Response to PCB3: The mERα biosensor displayed a quasi-linear response to PCB3 at lower concentrations (0-50 nM), with minimal variation between replicates. However, at higher concentrations (e.g., 5 µM), the response varied significantly, indicating that more experiments are required to establish a definitive dose-response curve.

6. Response to PAH: The mERα biosensor produced a strong response to benzanthracene, though the resulting dose-response curve lacked consistency due to high variability. This promising trend suggests that the biosensor can detect PAH, but further experimentation is needed to confirm its reliability.

7. Response to PAH in Milk: In the presence of milk, mERα's response to PAH decreased gradually as milk concentration increased. However, the signal was more stable compared to the BPA-milk experiments, with approximately a 50% reduction in signal intensity between water and milk fractions, suggesting that PAH detection is less affected by milk.

8. Response to Aroclor 1260: The mERα biosensor exhibited a dynamic range of 7000-11,500 luminescence units when detecting Aroclor 1260. However, the sensitivity within the tested concentration range was low, and the variation between replicates was high, making it difficult to draw definitive conclusions about the biosensor's effectiveness for Aroclor 1260 detection.

9. Response to Aroclor 1260 in Milk: The response of mERα to Aroclor 1260 in milk followed a step-like decline, with a sharp decrease in luminescence at higher milk fractions. This behavior was consistent with the other milk experiments, highlighting the significant influence of milk on biosensor performance. The variation in response patterns to different contaminants suggests that mERα's performance is context-dependent.

Composites

The Composites Table showcases the various composite parts that make up the functional modules of each biosensor device. Each composite part consists of a combination of promoters, receptors, or detoxification modules designed for specific detection or detoxification tasks. For example, BBa_K5477023 and BBa_K5477024 feature receptor modules tailored to detect PAHs, dioxins, and dioxin-like PCBs, while BBa_K5477027 and BBa_K5477028 are optimized for BPA detection through engineered estrogen receptor modules. Additionally, the composite parts BBa_K5477035 to BBa_K5477039 focus on detoxification, containing enzymes such as UGT2B15, UGT1A1, and CYP1A1 that metabolize harmful chemicals into less toxic or more easily excretable forms. These composite parts serve as the building blocks of the devices, providing the necessary functionality to detect and detoxify environmental contaminants.

Results

Parts

The Parts Table lists individual genetic components, including promoters, coding sequences (CDS), and regulatory elements, that make up the biosensor and detoxification systems. Promoters such as pRET2 and pSTE12 are used to drive constitutive or inducible expression of the receptor and detoxification modules, ensuring that the biosensors are responsive under the desired conditions. The table also includes various coding sequences for receptors like AhR, ARNT, and LexA-ERα, which are central to detecting endocrine disruptors like BPA and other toxic chemicals. Detoxification enzymes such as CYP1A1, UGT2B15, and CYP3A4 are also featured, responsible for metabolizing and neutralizing harmful substances. These individual parts allow the biosensors to be highly modular, enabling customization for different contaminants while maintaining efficiency and compatibility with standard assembly methods.

Results