Overview:
Synthetic biology has potential to address global problems associated with aquatic environments. Yet, few have been implemented in real-world aquatic systems. The lack of implementation 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 project addresses this knowledge gap with the following foundational advances that will help promote the design of safe, effective synthetic circuits for eventual deployment in real world aquatic environments.
Developed a Chassis Analysis Software Suite that Evaluates Chassis Survival in a Specific Aquatic Environment - Using Abiotic and Biotic (Consortia) Factors
In order to allow synthetic biologists to determine the feasibility of a chassis to survive in a specific aquatic environment, we developed a first-of-its-kind software that incorporates both community (consortia) composition and abiotic factors to predict survivability in an aqueous system of choice.
We created an accessible and extensible database, AQUERY, that includes over 2,000 metagenomic samples with corresponding environmental conditions. Unlike other databases of its kind, it includes processed taxonomic abundance data using metagenomic rather than 16S data which allows more accurate correlation between species presence and environmental factors.
The software (AQUIRE) utilizes an ensemble of several machine learning models that incorporates data from AQUERY to generate a species survivability score in an aquatic environment of interest and predict the feasibility of successful deployment. AQUIRE allows the user to input environmental data, which includes spatio-temporal features and nutrient levels, and an option for metagenomic information to determine the compatibility of a chassis in that environment.
Together, these software tools provide synthetic biologists with resources that inform chassis selection for more reliable implementations in real-world environments.
Predictive Mathematical Model to Determine the Parameters that Would Allow for a Successful Deployment on an Engineered Chassis
We developed a modular mathematical framework that allows synthetic biologists to assess and approximate the parameters which are necessary for the successful deployment of an engineered chassis in a diverse aquatic environment. This framework is based on the transport equations and rheological concepts, which can be expanded to implementation of SynBio chassis in a wide range of fluid dynamic environments. It parameterizes a value determined by our survivability predictive software AQUIRE into the mathematical modeling framework in order to incorporate the biotic and abiotic factors that contribute to the chassis functionality.
Utilizing this framework, we created novel differential equation models tailored to three distinct case studies: remediation of freshwater algal blooms, biofilm removal in household plumbing, and corrosion prevention in marine settings, which are all accessible to researchers in the field pursuing these specific applications.
Development of Novel Engineering Solutions to (1) Corrosion Using Engineered Bacillus subtilis and (2) Detrimental Biofilms in Household Plumbing Using Mycobacteriophage
We developed novel proof-of-concept engineering solutions to address pressing problems in dynamic aquatic environments. First, we developed different biofilm regulatory pathways to prevent corrosion on steel and successfully utilized phage as a remediation strategy for mycobacterial biofilms in pipes. We have, for the first time, validated the influence of Bacillus subtilis on steel corrosion behavior in a non-sterile marine microcosm and identified crystal structures that have not been previously reported in the literature. These findings demonstrate that B. subtilis possesses the ability to modulate corrosion processes in natural environments, offering new directions for corrosion-prevention research. In addition, we novelly conducted transcriptomics analysis on B. subtilis gene expression in a marine microcosm, revealing the distinct transcriptional patterns between laboratory and natural conditions.
Second, we have also demonstrated that a specific cocktail of mycobacteriophage demonstrates potential to remove biofilms in household plumbing. While these solutions show promise, both these engineering solutions require further investigation and experimentation in order to be fully effective strategies. Our foundational work has demonstrated that chassis behave significantly different in real-world environments and must be engineered to account for these differences.
RNA-Seq Meta-Analysis and Design Principles
We developed design principles based on comprehensive meta-analysis of RNA-Seq datasets mined from the literature to provide insight into more reliable circuit design for real-world implementation. By systematically identifying differentially expressed genes and pathways between laboratory and natural conditions, we addressed a major knowledge gap that has limited the translation of synthetic biology from lab to field. Our contribution extends beyond traditional bioinformatics: our novel hybrid pipeline integrates AI-assisted analyses (Copilot, Gemini, ChatGPT) with established computational workflows, paired with rigorous vetting to ensure reliability.
This framework produced a curated, comprehensive resource of expression patterns and pathways that future teams can potentially use to inform chassis engineering and circuit design in realistic environments. We created a usable guide on how to generate design principles for a specific chassis and application of interest for synthetic biologists to utilize in their engineering design.
Developed a Comprehensive Series of Measurement Strategies (and accompanying guides)
Assessing the aquatic deployment potential of engineered constructs and chassis requires a suite of controlled measurement techniques for comparing chassis behavior and persistence in simulated real-world versus sterile in vitro conditions. We developed a comprehensive framework for measuring elements of chassis functioning at different levels of measurement accessibility and complexity, ranging from visual assessment to microscopy and total transcriptomic analysis.
Our techniques provide a diverse toolkit that other research groups wishing to test their SynBio devices may selectively draw from to accommodate their resource availability and specific research needs. In order of increasing complexity, our measurement techniques included:
- Macroscopic visual analysis: We developed a basic visual analysis system for assessing M. aeruginosa cyanophage presence in host infection assays. We also developed a unified set of wetlab procedures for conducting cyanophage assays and recording visual assessments of phage presence over an extensive time course. To assess chassis persistence over time under simulated real-world aquatic conditions, we developed a system for conducting semi-automated colony counts by prompting AI chatbots for assistance with visual analysis and colony identification.
- Microscopy: We developed atypical scanning electron microscopy and digital microscopy techniques to evaluate the effectiveness of our chassis strains and novel engineered constructs of interest for multiple aquatic applications
- Molecular analysis: We developed procedures for collecting growth curve measurements based on molecular fluorescence to assess and compare the growth and persistence of specific engineered and non-engineered bacterial strains under different conditions.
- Genomics and transcriptomics We generated large, well-controlled metatranscriptomic datasets containing RNA sequence data for chassis and total microbial communities under analogous simulated real world and controlled in vitro conditions. We generated parallel 16S DNA sequence datasets that provide species abundance measurements at the same timepoints and conditions as those used for RNA sequencing. Our analyses revealed differential gene expression and variation in chassis abundance across microcosm and control groups, indicating that engineered bacteria function and persist differently when deployed in realistic environments as compared to in vitro conditions. Importantly, we developed a pipeline for others to conduct a similar analysis.
- Bioinformatic data mining:We included a type of measurement not typical for iGEM teams, but increasingly important for thorough measurement in projects—namely bioinformatic mining of existing data useful to our project. We conducted a meta-analysis of existing RNA sequencing datasets, a largely unexploited source of gene expression data that would yield relevant patterns in bacterial behavior and functioning under conditions associated with deployment in aquatic environments. We also developed a system for harnessing AI tools in order to access relevant studies and data for meta-analysis.
Developed User-Friendly Hardware Solutions for Testing Engineered Constructs in Real-World Environments
To close the gap between synthetic biology design and real-world application, we developed cost-effective, reproducible, and modular hardware systems that simulate realistic aquatic environments. These platforms enable testing of engineered biological chassis under fluid dynamic conditions that closely mimic nature. We constructed three adaptable microcosms representing seawater ecosystems, freshwater lake environments, and household plumbing systems. Each model is designed for versatility, allowing researchers to evaluate a wide range of synthetic constructs in context-specific conditions. This approach enhances reliability, scalability, and accessibility—bringing synthetic biology closer to practical deployment.
Development & Deployment of New Educational Tools and Guidebook (and Quick Guide) for Getting Started in SynBio in Aquatic Systems
We led a series of innovative education initiatives to make synthetic biology accessible and engaging for the broader community. Through interactive workshops, public talks, and partnerships with local schools and science organizations, we introduced core concepts of synthetic biology and AI in biology to diverse audiences—including elementary students. We developed novel teaching tools, custom lesson plans, and visual materials to explain complex topics like phage enrichment and biofilm dynamics in approachable ways.
To extend our impact beyond local outreach, we created a guidebook for researchers that includes strategies for applying design principles to improve circuit reliability in aqueous environments, scalable measurement techniques, and instructions for using our novel software and AQUERY database to inform chassis selection.