Problem:
The flow of water is essential to life on Earth. It distributes nutrients, creates and maintains ecosystem biodiversity, enables agriculture through irrigation, removes waste products, regulates temperature, supports the planet’s water cycle, and provides a source of clean energy. However, water flow can have profound negative consequences, which are often exacerbated by human interaction (Plessis, 2022). The same flow that can replenish a drying lake or provide running water to a household can also contaminate an ecosystem with pollutants, shuttle pathogenic bacteria through a showerhead, erode shorelines, and degrade habitats.
Synthetic biology offers potential to address these problems. Yet, few have been implemented in real-world aquatic environments. This is largely due to the significant gap in foundational knowledge of how engineered constructs function in real-world environments, which leads to insufficient safety understanding, which in turn results in a lack of effective policy. In aquatic systems, chassis are subjected to diverse community dynamics, fluctuating nutrient and mineral concentrations, and physical forces, which affect how organisms behave in their respective environments.
Our Solution:
We address this gap in foundational knowledge through a combination of predictive software development, dynamic modeling, and testing of novel constructs in simulated real-world environments using a wide array of measurement techniques, from macroscopic visual assaysto RNA-Seq analysis. Our project generates novel insights into construct and chassis behavior in aquatic systems, identifies design principles for robust water-based synthetic circuits, and provides an array of resources to support innovation in aquatic synthetic biology.
AQUERY and AQUIRE: Novel Software Tools for Chassis Survival Analysis
Selecting an appropriate chassis is essential for applying synthetic biology solutions in aquatic systems. However, deployment in real-world aquatic environments is often halted by the stark difference between controlled lab and complex field conditions. Predicting a chassis's survival is extremely difficult due to the lack of foundational knowledge and resources that integrate the complexities of a real-world aquatic environment. To directly address the lack of knowledge in chassis survivability in aqueous environments, we created the novel software tools AQUERY and AQUIRE. These tools are designed to inform users on chassis survival in a chosen deployment location, serving as a critical verification step to confirm feasibility before putting in an excessive amount of resources into a construct that may not be compatible with the environment in which it is meant to be eventually deployed.
AQUERY is a first-of-its-kind database that incorporates both metagenomic data and abiotic conditions such as temperature, nutrient levels, and pH. In order to provide more accurate insight into the community dynamics of aquatic systems, AQUERY incorporates quality-controlled, processed taxonomic abundance data from metagenomic (rather than 16S) samples to yield accurate taxonomic data.
AQUIRE is a unique environmental and metagenomics-based machine learning model, which leverages the AQUERY dataset to predict chassis survival in a chosen environment. AQUIRE outputs survivability scores based on the user input of a selected chassis and information on the abiotic factors of the deployment environment and/or species abundance.
Both AQUERY and AQUIRE serve as streamlined pathways that offer guidance on chassis selection for more reliable implementation in real-world aquatic systems, offering a toolkit to allow more informed chassis deployment.
Case Study Investigations Comparing Laboratory vs. Simulated Real-World Aquatic Environments
In order to investigate differences in chassis behaviour in laboratory conditions compared to real world environments, we chose three case studies, each in a vastly different aqueous system to explore a wide range of fluid dynamic conditions. These case studies provide insight into the disparities of chassis functionality and efficiency between lab conditions and real-world environments.
Aquatic Case Study I: Microbial Induced Corrosion Inhibition in Marine Environments
Oceans face persistent issues with metal corrosion, which has become a critical global concern. Microbial-Induced Corrosion Inhibition (MICI) presents a promising and sustainable approach to combating corrosion. Bacillus subtilis forms strong and robust biofilms, providing a suitable chassis for MICI. However, while natural Bacillus subtilis strains are capable of forming strong biofilms, they often lose key regulatory traits during domestication—particularly those involved in biofilm formation. Lab strains such as B. subtilis 168, though genetically competent, have lost the ability to secrete extracellular polymeric substances (EPS), which are essential for biofilm development (McLoon et al., 2011). We developed circuits with different biofilm regulatory pathways to test which would be most efficient in the biofilm formation of the domesticated B. subtilis strain.
We engineered B. subtilis strains to have enhanced biofilm-forming capabilities by upregulating EPS-related genes. Using the competent but biofilm-deficient lab strain B. subtilis 168, we tested multiple regulatory pathways to identify those most effective at promoting robust biofilm formation and corrosion resistance in real-world environments. These included the SinI/SinR regulatory pathway, including downstream components as TasA protein and the TapA-SipW-TasA operon, as well as the expression of BslA protein to determine their effects on biofilm thickness and strength
We tested the corrosion-prevention abilities of both the engineered constructs and the natural strains in seawater microcosms to evaluate chassis behavior differences under lab conditions and in more realistic environments. This approach also provided valuable insights into the functional differences between engineered and natural strains, particularly in their ability to perform in real-world environments.
Aquatic Case Study II: Freshwater Harmful Algal Bloom (HAB) Remediation
The cyanobacterium Microcystis aeruginosa is a leading contributor to harmful algal blooms (HABs), which drive significant biodiversity loss in freshwater ecosystems and result in toxin release that is harmful to humans (Bhatt et al., 2023). Synthetic biology tools may offer a variety of potential M. aeruginosa HAB treatment strategies.
To clarify the effect of simulated real-world environmental conditions on the effectiveness of SynBio tools for M. aeruginosa HAB remediation, we investigated two SynBio-based HAB treatment strategies
- We investigated the feasibility of HAB treatment with engineered cyanophage, viruses that infect cyanobacteria and are promising because of their natural host-specificity and self-propagating nature (Grasso et al., 2022; Bhatt et al., 2023; Aranda et al., 2023). Despite their HAB-remediation potential, few M. aeruginosa cyanophages have been isolated (Guo et al., 2024). We developed protocols for isolating novel cyanophages and found evidence of a putative M. aeruginosa cyanophage from an environmental sample. We concluded that strong host defense systems, among other factors, may limit the effective deployment potential of cyanophage for HAB remediation.
- We investigated the feasibility of HAB treatment with Acinetobacter baylyi ADP1, a highly competent and engineerable chassis bacterium that possesses natural algicidal capabilities (Li et al., 2016, Yi et al., 2015, Suárez et al., 2017). To assess the A. baylyi’s real-world deployment potential, we analyzed the chassis’ persistence and gene expression in flowing lakewater microcosms as compared to static control co-cultures. Our data indicate that A. baylyi exhibited lower survival and different patterns of gene expression in realistic microcosms as compared to control cultures.
Our results demonstrate that complexities associated with real-world environments may influence the effectiveness of SynBio-based HAB remediation solutions. Synthetic biologists must anticipate real world complexities when designing and testing HAB remediation tools.
Aquatic Case Study III: Phage Treatment of Biofilms in Household Pipes
Daily exposure to nontuberculous mycobacteria (NTM) is an increasing global concern, as these bacteria contribute to pulmonary and extrapulmonary diseases and are implicated in lung and breast cancer-associated microbiomes (Maranha 2024). A primary source of human exposure to NTM is household water systems, where mycobacteria form resilient biofilms (Dowdell, 2019). Mycobacteria, in particular, form robust, disinfectant-resistant biofilms that contribute to increasing risks of pulmonary infections.
Phage have shown promise as an alternative solution to target bacterial biofilms (Tian, 2021). To exploit the evolutionary adaptations of bacteriophages developed through their co-evolutionary dynamics with bacterial hosts, we formulated a phage cocktail aimed at maximizing biofilm disruption and bacterial lysis efficiency. The selected phages were chosen based on their genetic characteristics, lytic capabilities, and potential for biofilm interference.
In order to determine if phage would provide a suitable treatment strategy for biofilms in household pipes, we testing the efficiency of phage to lyse bacteria both in lab conditions and in simulated real-world environments. We did so by constructing microcosms that mimic household PVC pipes and monitoring biofilm presence and structure before and after phage treatment.
AQUAINT: A Modular Mathematical Framework for Aquatic Implementation Prediction
To allow deployment of engineered systems in aquatic environments, there needs to be a predictive mathematical model that provides under which conditions and parameters a chassis would be able to function in fluid dynamic environments.
We created a modular framework based on the transport equations and rheological concepts that outlines design principles for differential equation models for SynBio implementation in aquatic systems. This framework also incorporates parameters based on chassis survivability scores outputted by our software, AQUIRE.
Utilizing our framework, we designed partial and ordinary differential equations to model our three aquatic case studies. Each model captures key aspects of fluid transport, biological persistence, and interaction dynamics, offering a foundation for predictive design and deployment. These models allow us to identify the key design principles for genetic engineering and effective treatment, guide the development of constructs, and evaluate how well a given chassis can function within its specific aquatic environment.
RNA-Seq Meta-Analysis to Identify Overarching Design Principles for Aquatic Synthetic Biology Solutions
In addition to using three case studies to contribute to foundational knowledge of how engineered chassis perform in real world environments, we also mined the potential wealth of information available in existing databases. We employed AI and our own RNA-Seq analysis pipelines to perform a comprehensive meta-analysis of RNA-Seq data mined from the literature and available databases. We identified commonly differentially expressed genes and their associated pathways between laboratory and natural conditions. This allowed us to extract key design principles for circuits in order to allow – eventually — for the safe and effective deployment of synthetic constructs in real-world aquatic environments.
Inspiration:
Our project is inspired by local problems that affect our university and geographic region. The surrounding Virginia coastal systems—which include the eastern shore, various wetlands, tidal marshes, and the Chesapeake Bay—equip us with firsthand experience of the need for advancement in the methods and tools used to handle contaminated water flow due to recurrent issues. For example, there are water quality crises in Richmond and the Hampton Roads area, frequent algal blooms across Virginia, and excessive coastline erosion in the broader Chesapeake Bay Watershed.
William & Mary has officially declared 2025 to be the Year of the Environment and plans to begin sustainability initiatives both locally and outside of the William & Mary community. Furthermore, William & Mary has chosen water as a key area of focus—finding ways to improve and ensure the resilience of water systems–an endeavour that shaped the trajectory of our project.