Project Description
Water pollution in Mexico

Water Pollution in Mexico

💧 Current Situation

In Mexico, access to enough safe, acceptable, and affordable water for personal and domestic consumption is a human right established in the country's Political Constitution (Fondo para la Comunicación y la Educación Ambiental, 2021). However, this need has not been met for most of the population. National statistics state that 12 million people in Mexico currently lack access to safe drinking water, mainly due to contamination of water bodies (Toche, 2023).

With a total registered population of 126'705,128 people in 2021 (The World Bank, 2022), that means that over 9.47 % of the population lacks this basic human right.

Approximately 1 in 10 Mexicans do not have access to safe drinking water.

According to information from the National Water Quality Measurement Network (Renameca), 59.1% of the rivers, streams, lakes, lagoons, dams, and coastal areas are polluted (Rodríguez, 2022). Every year, millions of cubic meters of wastewater as well as municipal, industrial, and agricultural residue, including emerging contaminants, are inappropriately treated or untreated and discharged into bodies of water. Water pollution has a severe impact on ecosystems and health (Fondo para la Comunicación y la Educación Ambiental, 2021), causing approximately 95,000 deaths in children under the age of five (Aristegui Noticias, 2019).

Figure 1. Water quality indicators in water sources of Mexico. If quality results indicate that one or more criteria are not met, the site is painted red or yellow. Those in green comply with the national standard (CONAGUA, 2022).

❗ Contaminants of Interest

Emerging contaminants are recently identified categories of unregulated pollutants found in surface and groundwater. These include, but are not limited to pharmaceuticals such as erythromycin, personal care or household cleaning articles, agricultural products, among others. These products typically contain bioactive components and possess bioaccumulative properties, meaning they can be widely distributed and hard to break down,(Water Resources Mission Area, 2019).

In Mexico, there is limited research on the presence of chemical pollutants in bodies of water and their potential environmental risks. There is a lack of simple methods for detecting and quantifying these emerging contaminants, including erythromycin and cadmium. Current detection techniques such as chromatography and extraction methods are expensive, time consuming, and require trained personnel (García-Corcoles, 2018). Communities near these polluted water bodies do not have access to the economic and technological resources to access these techniques.

Due to the lack of inexpensive and easy to use detection methods, our team decided to create a biosensor able to detect erythromycin and cadmium in contaminated bodies of water.

Our Approach: The EC-FRET Biosensor

🧐 What is our Biosensor?

Our project aims to develop an inexpensive and highly efficient biosensor in a biological platform (E. coli BL21), capable of detecting emerging contaminants, under the name of “The EC-FRET Biosensor”, with “EC” (read as “easy”) representing Emerging Contaminants. This biosensor consists of a fusion construct made up of two fluorophores, Enhanced Cyan Fluorescent Protein (ECFP) and mVENUS, and a detector enzyme, EryK in the case of erythromycin and a phytochelatin synthase (AtPCS) to detect cadmium.

To design this biosensor, we developed a profile based on user requirements, with a strong emphasis on accessibility. Our goal is to create a portable device that can be easily operated by anyone to analyze water samples and determine the presence of emerging contaminants. To meet these criteria, we developed a hardware device that has the function of detecting and quantifying the fluorescence of the presented FRET construct through calibrations and mathematical models.

❓ How does it work?

FRET (Förster Resonance Energy Transfer) is a phenomenon involving two chromophores, typically fluorophores. It occurs when energy is transmitted from a donor to an acceptor over a distance, with the excited electron donor transferring energy to an acceptor molecule.

In our system, the donor fluorophore is ECFP, while the acceptor fluorophore is mVENUS, a yellow fluorescent protein. Additionally, we intend to incorporate AtPCS into the FRET system, replacing EryK and linking it with ECFP and mVENUS.

In our construct, the enzyme EryK serves as a sensor for erythromycin. When erythromycin binds to EryK, it induces a conformational change in the system. This leads to the movement of the fluorescent proteins, altering the distance between them which leads to a change in the FRET signal. In short, the transition from a ligand-unbound to a ligand-bound conformation causes the distance between the fluorophores to shorten, enabling energy transfer to occur, and therefore fluorescence of the acceptor fluorophore to occur.

Figure 2. Schematic diagram of a single molecule FRET system in the presence of a substrate such as erythromycin or cadmium.

💡 What inspired us?

Our inspiration for developing this biosensor arises from an inspirational transformative visit to the Santiago River in Jalisco, Mexico. Not only is it known as the most polluted river in Mexico (Martínez-González, 2009; Revivamos el Río Santiago, 2023), but it is also connected to the main water source for Guadalajara and surrounding areas (CEA, n.d.), Lake Chapala.

As we gazed at the turbid and putrid waters on the shores of the Santiago River, it became abundantly clear that water pollution was not just an abstract textbook topic, but a harsh reality affecting the lives of countless people. Once full of life and a source of livelihood for the communities, the river is now a dangerous water source affecting both the residents and the surrounding ecosystem.

This experience was eye-opening and profound, as it not only showed us the severity of the water pollution problem, but also brought us face to face with its tangible and devastating effects on the environment and the communities living nearby. As we reached out to local residents and listened to their stories and concerns, it became clear that there was a pressing need for a solution that could empower them to monitor water quality themselves. People shared their fears about the invisible threat of emerging contaminants and the lack of access to reliable and affordable detection methods. This first-hand encounter validated our conviction about the need for our biosensor project.

As we embarked on the journey of biosensor development, we carefully considered the pollutants to target in our first version. We weighed various factors, such as the presence of different contaminants in water, adverse reactions from consuming them, enzymes involved in their detection and their kinetic parameters. After extensive research and deliberation, we chose erythromycin and cadmium as the contaminants to detect.

Erythromycin is an antibiotic that can find its way into water sources through the incorrect disposal of pharmaceutical waste and is a concern due to its potential effects on aquatic ecosystems and human health such as antibiotic resistance (Huovinen et al., 1997). On the other hand, cadmium is a heavy metal often released into water as a result of industrial processes and can have severe environmental and health implications such as kidney failure (Bernard, 2008).

The first iteration of our biosensor will prioritize the detection of these contaminants, with the aim of delivering a valuable tool to assess and monitor water quality. By doing so, we hope to contribute to the mitigation of water pollution issues and promote informed decision-making regarding the use of water resources in Mexico and beyond.

We are committed to making a positive impact by providing accessible tools for water quality assessment and promoting responsible management of our precious water resources.

🌍 Other Biosensors

In the process to develop our FRET biosensor, it was important to review existing technologies, methods and related studies to validate the feasibility and potential of our idea.

In recent years, the development of biosensors based on FRET technology has experienced significant progress, opening up exciting possibilities for the detection of various molecules in different contexts (Chen et al., 2022; Limsakul et al., 2018; Zhang et al., 2019). In order to validate the concept of a FRET biosensor for detecting cadmium and erythromycin in water samples, we can draw inspiration and knowledge from the following studies.

  • In one study, FRET-based biosensors were designed to quantify cellular concentrations of 2-oxoglutarate (2-OG) in Escherichia coli and Anabaena sp. These biosensors showed fast and accurate responses to varying levels of 2-OG, highlighting the potential of FRET-based biosensors for continuous real-time monitoring of target molecules, especially in response to environmental changes (Chen et al., 2018).
  • Another innovation, an optical biosensor for leucine-isoleucine-valine (OLIVe), was developed to detect branched-chain amino acids (BCAAs). The ability of OLIVe to establish a reliable correlation between FRET signals and intracellular BCAA concentrations in individual living cells demonstrates the potential of FRET-based biosensors to provide quantitative information about target molecules in complex cellular environments (Yoshida et al., 2019)

Furthermore, FRET-based analytical methods have gained widespread acceptance for detecting food contaminants. Their adaptability, specificity, and efficiency make them practical tools for the rapid and reliable identification of contaminants in food samples (Liang et al., 2023).

To conclude, the development and application of these FRET-based biosensors in various biological and environmental contexts demonstrate their potential as versatile tools for molecular detection. These examples highlight the possibility of applying a FRET-based biosensors in our own concept, allowing us to move forward in our development efforts and contribute to addressing the critical problem of water contamination.

Conclusion and Future Plans

The biosensor proposed by the team offers a practical means of detecting emerging contaminants present in water bodies, such as erythromycin and cadmium. This innovative approach represents a cost-effective and straightforward solution for identifying these pollutants, which did not exist before. Consequently, this technology holds the potential to empower underserved communities residing in proximity to polluted water sources by enabling them to assess water quality and ensure that products made by manufacturing companies don't pose a risk to long-term health. This can aid in averting various health issues and even fatalities associated with exposure to such contaminants.

To validate the efficacy and sensitivity of our biosensor, it is essential to carry out tests involving a range of erythromycin concentrations. These experiments will serve a dual purpose: first, to assess the biosensor's fluorescence response through the device, and second, to gain insights into the behavior of the FRET system. While time constraints prevented us from conducting these tests within our present scope, it is strongly suggested to perform them in the future to optimize the performance of the biosensor.

As for our future projections, we aspire to enhance the detection capacity of our system to cover a broader spectrum of contaminants. Furthermore, we remain committed to ensuring accessibility to both local communities residing near water sources and specialized water quality monitoring entities, all under the name of The EC-FRET Biosensor. For further details, we invite you to explore the Entrepreneurship page.

FRET-eryK & AtPCS

The goal of this project is to produce a biosensor that detects emerging contaminants such as pharmacological components or heavy metals. Once our construct binds to the pollutant, conformational changes within the system will lead to a detectable FRET signal. In our case, the target pollutant is erythromycin.


FRET

Förster Resonance Energy Transfer (FRET) is a a measurable, physical phenomenon between two chromophores, often fluorophores, in which energy is transferred from a donor chromophore to an acceptor chromophore in a through-space, distance-dependent manner (Lim et al., 2022; Algar & Krause., 2022). FRET measurements between two fluorophores, given that their characteristic FRET distance is known, can be used to detect conformational changes in biomolecular systems. FRET occurs when an excited electron donor transfers energy to an acceptor molecule, which causes it to relax to the lowest excited state. Due to energy conservation laws, lost energy needs to be transformed. In the absence of an acceptor fluorophore, this transpires as the emission of a photon. If the acceptor fluorophore is close enough proximity, the donor will transfer its energy to excite the acceptor (Suppan, P., 1994; Yang et al., 2022).


Enhanced Cyan Fluorescent Protein

The donor fluorophore in our system is Enhanced Cyan Fluorescent Protein (ECFP). This protein is related to Green Fluorescent Protein (GFP), originally isolated from the bioluminescent jellyfish Aequorea victoria, and it only differs by a few mutations. The main mutation responsible for the excitation and emission spectrum shift from GFP to ECFP is Y66W (Mérola et al., 2014; Kremers et al., 2006). This substitution replaces the phenol group with an indole which alters the fluorescent properties of the conjugated system. Additional mutations were introduced to the β-barrel that surrounds the central chromophore to rescue the original fluorescence intensity (Heim & Tsien, 1996; Rizzo et al., 2004).

The excitation spectrum for the fluorophore shows one peak at 435 nm, corresponding to blue/cyan visible light, and the emission spectrum has a peak at 476 nm (Figure 1).

Figure 1. Emission and excitation spectra for ECFP (Lambert, T. J., 2019).

Erythromycin C-12 hydroxylase (EryK)

This enzyme is a cytochrome P450 from Saccharopolyspora erythraea and is responsible for C12 hydroxylation of erythromycin B and D (Lambalot et al., 1995; Gaisser et al., 1997).

Within our fusion construct, EryK acts as a sensor for erythromycin which, upon binding, will undergo a conformational transition. This should alter the distance between the fluorescent proteins resulting in a change in a FRET signal (Kim et al., 2016).

Figure 2. Morph of eryK in an open conformation (PDB ID: 3ZKP) to a closed conformation (PDB ID: 2JJN).

Both open and closed structures have been previously determined through X-ray crystallography. Using these it was possible to create a morph between these structures to visualize the conformational change between these two states (Figure 2). The existence of conformational differences between the apo- and holo-states of eryK is promising, and we intend to leverage these motions to alter the distance between the fluorophores, and detect erythromycin binding via changes in the FRET signal.


mVENUS

The acceptor fluorophore present within our system is mVENUS, a yellow fluorescent protein derived from avGFP (Kremers et al., 2006; Lee et al., 2020).

The excitation spectrum for the fluorophore shows a singular peak at 515 nm, and the emission spectrum has a peak at 529 nm (Figure 3).

Figure 3. Emission and excitation spectra for mVENUS (Lambert, T. J., 2019).

The goal of this project is to generate a new biopart to incorporate into the FRET system for heavy metals detection. Once our construct binds to the pollutant, conformational changes within the system will lead to a detectable FRET signal. In our case, the target pollutant is cadmium.


Introduction to AtPCS1

The enzyme chosen for the generation of the biopart was phytochelatin synthase (PCS; EC 2.3.2.15), which catalyzes the synthesis of glutathione (GSH) polymers called phytochelatins (PCs) (Figure 1). These are produced as a mechanism of resistance and accumulation in algae, yeast, plants, and worms in response to heavy metal stress (García-García et al., 2014; García-García et al., 2020; Grill et al., 1985). Phytochelatins bind to metals intracellularly and render them inactive. This is because the substrate of PCS is GSH and other metal-bis-glutathione complexes (García-García et al., 2014; García-García et al., 2020; Schmöger et al., 2000). Additionally, depending on the species being studied, the enzyme requires the presence of specific heavy metals , with cadmium being the main metal ion cofactor in plants and other organisms. The complexes formed by the metal and PCs can be subsequently compartmentalized into vacuoles, chloroplasts, and mitochondria (García-García et al., 2014; 2020).

The PCS enzyme from the model organism Arabidopsis thaliana (AtPCS1) was selected for the creation of the biopart (BBa_K4763007) due to its more extensive characterization in the synthesis of phytochelatins (García-García et al., 2014; García-García et al 2020; Mendoza-Cózatl et al., 2005).

Figure 1. Phytochelatin synthesis from GSH, catalyzed by PCS (based on García-García et al., 2016). .

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