Progress 07/01/24 to 06/30/25

Outputs
Target Audience:The target audiences reached during this reporting period included graduate and undergraduate students specializing in environmental engineering and synthetic biology at CU Boulder, wastewater treatment professionals from municipal utilities involved in collaborative workshops, and researchers focused on synthetic biology and emerging environmental contaminants. Additionally, regulatory agencies responsible for environmental regulation oversight and broader community members were indirectly engaged through outreach and informational dissemination activities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?STRATAGEM provided significant professional development opportunities for the two graduate research assistants (GRAs) and the two undergraduate researchers involved. GRAs received intensive training in advanced synthetic biology methods, molecular genetics techniques, computational genomics, and bioinformatics tools. Specifically, GRAs learned plasmid design, Nanopore sequencing techniques, batch and biofilm reactor setup, and microbiological assay methodologies. Additionally, team members enhanced their computational and software engineering skills through developing bioinformatic pipelines using Python and R. Undergraduates were actively trained in basic laboratory procedures, molecular techniques, and assisted in biofilm and hydroponic system operations, providing hands-on experiential learning. All trainees presented their research progress at internal lab meetings and national conferences, fostering presentation skills, scientific communication, and collaborative teamwork. How have the results been disseminated to communities of interest?The results of STRATAGEM are gaining positive interest from the community after presentations at multiple academic conferences by PI Mansfeldt and GRA Taylor Cason. Additionally, PI Mansfeldt organized two workshops over the past year. Oral Presentations Keynote Talk. International Water Association 11th Microbial Ecology and Water Engineering Conference. "Predicting and tracking the environmental range of synbio modified microorganisms". June 4, 2025. Atlanta, Georgia, USA. Poster Presentations American Society of Microbiology. "Integrating DNA Watermarks into Genetically Engineered Microorganisms to Support Agricultural Runoff Assessments." Presented by GRA Taylor Cason. June 20, 2025. Los Angeles, California, USA Built Environment Microbiome Gordon Conference. "The Need for Practical Prediction and Tracking of Emerging Synthetic Bioproducts in the Environment". June 26, 2025. Waterville Valley, New Hampshire, USA. Workshop and Session Organization Co-Organized Conference Session. Association of Environmental Engineering and Science Professors 2025 Research and Education Conference. "Preventing Good Microbes from Going Bad: Environmental Biotechnology Applications and Management in the Era of Synthetic Biology." May 22, 2025. Durham, North Carolina, USA Organized Conference Workshop. International Water Association 11th Microbial Ecology and Water Engineering Conference "Ethical Dimensions of Synthetic Microorganisms and Communities in Microbial Ecology." June 2, 2025. Atlanta, Georgia, USA. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will focus on advancing each project objective from foundational development into active experimentation and validation phases. Specifically: Objective 1: We will utilize our established barcode-sequencing pipeline to begin systematic evaluation of barcode stability in simulated environmental scenarios. Objective 2: We will initiate batch-suspended media degradation tests using our validated qPCR and sequencing protocols, quantifying the persistence and degradation kinetics of barcoded organisms. Objective 3: With the biofilm reactor fully operational, we plan to conduct initial biofilm growth experiments, characterizing barcode persistence and tracking organism viability over extended growth periods. Objective 4: We will operate the fully assembled agricultural and wastewater testbeds continuously, analyzing microbial community dynamics, barcode dissemination, and environmental persistence. Objective 5: The ecological threat assessment computational framework will be structured, focusing on integrating empirical results from Objectives 1-4 to inform practical management strategies.

Impacts
What was accomplished under these goals? Objective 1. Develop and test genetic watermarks to enable mark-recapture of GEMs during threat assessments and field deployments Accomplishment. First, we have developed the computational pipeline to identify unique barcode sequences that can be utilized in tagging modified organisms. This pipeline has been developed within Python and considers the entire complete genomes available at the National Center for Biotechnology Information. The code searches for and demonstrates that the short barcode is unambiguous in identifying the modification as an anthropogenic addition. Second, we have optimized the selection and design criteria for incorporating those barcodes identified computationally into test organisms. When ordering test plasmids, we identified that (1) certain motifs within the computationally identified barcodes must be avoided and (2) integrating the barcode structure must be designed in a "codon frame" (i.e., having the final nucleotide addition divisible by three). Following these two primary rules, we successfully incorporated the barcode on a plasmid with an antibiotic resistance marker. Third, we successfully incorporated that barcoded selective plasmid into E. coli. E. coli was thereafter able to grow with the barcode present and on the appropriate selective media, indicating that the barcode did not halt the function of the plasmid. Furthermore, after repeated culturing of E.coli, the incorporation did not alter the growth patterns when compared to the equivalent unbarcoded bacteria. Fourth, sequential Nanopore sequencing also revealed that the barcode was stable over these successive generation growth curves. Throughout these growth conditions, the barcode neither mutated to a high degree nor altered in frequency when compared to other genomic elements. We have additionally established the sequencing pipeline, everything from the within-laboratory wetlab extraction, labelling, and sequencing of the barcoded organisms to the bioinformatic pipeline utilized to retrieve the barcodes from the mixed-community data. Overall, developing and ground-truthing this barcode is a critical milestone that enables Objectives 2-5. This accomplishment provides a robust, field-deployable genetic marker, advancing our ability to reliably track genetically engineered microorganisms in realistic environmental contexts. Objective 2. Establish batch-suspended media degradation tests similar to chemical regulatory risk assessments to inform biological threat assessments Accomplishment. We have developed the growth, extraction, and sequencing protocols for tracking the barcoded organisms within batch assays. Additionally, the GRA on the project designed the barcodes in such a manner to allow for targeted qPCR assays. These qPCR assays will be utilized to monitor the persistence and degradation of the barcode and furthermore compliment the sequencing runs. Full qPCR has been run on standard control DNA, demonstrating that the technique works and is available for analyzing the batch growth results. Protocols and assays are now fully developed and validated, positioning us for batch growth experiments scheduled for the coming reporting period. Objective 3. Establish batch attached-growth biofilm degradation tests. Accomplishment. We have purchased, setup, and established the biofilm flow device. We additionally have established the pump flow network to deliver either the mock river or wastewater to the flow reactor. Furthermore, this biofilm reactor enables swapping and comparing different biofilm growth strata that we will utilize in future assays. We have therefore completed the initial construction of the biofilm reactor system. This prepares the groundwork for initiating experimental biofilm degradation tests in the next phase of the project. Objective 4. Establish a model agriculture runoff cycle (combination of sewer, activated sludge, and stream systems) to test hazard assessments Accomplishment. First, we designed and constructed the pipeflow network representative of hydroponic agricultural systems. These testbeds connect LED-illuminated hydroponic growth racks with a pipe network that we will now run continuously to establish a stable microbiological community. We currently have six completed hydroponic systems established within a series of biocontrol rooms. The outflow of each system will connect with a pipe-rack to simulate the wastewater cycle. These systems overall have been economical to develop and straightforward to operate. Second, we have designed and assembled the materials for the test agricultural fields. These fields have been planned to capture parallel soil systems that drain into a model creek network. These testbeds will be watered through an overhead spray network that we designed to simulate rainfall. Third, we have established relationships with local Water Resource Reclamation Facilitiesto provide activated sludge for our model wastewater treatment reactors. Each of these testbeds have been constructed in a 2,400 square foot dedicated water pilot device exploration space. This facility expansion has been provided by the University of Colorado Boulder in support of this project. All major infrastructure and testbed setups for agriculture and wastewater cycles have been completed. Systems are near fully operational, allowing transition into long-term simulation studies in the upcoming period. Objective 5. Develop a framework for utilizing this ecological threat assessment technique to inform the management and regulation of GEMs. Accomplishment. We have established the computational architecture within GitHub and have identified the primary GRA that will focus on advancing this objective. We will next focus on developing the detailed ecological threat assessment framework.

Publications

Progress 07/01/23 to 06/30/24

Outputs
Target Audience:The main target audience reached during this reporting period were other scientists and practicing micorbiologists. The graduate student researcher presented a poster covering the initial findings surrounding the framework of the established genetic system to establish the mutant strainsat the 2024 American Society of Microbiology in Atlanata, Georgia June 19-23, 2024. Additionally, PI Mansfeldt presented an overview of the framework as a poster at the Gordon Research Conference:Water meeting in Holderness, New Hampshire June 23-28, 2024 to an audience of primarily other environmental micorbiologists and chemists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The main training and professional development activities resided around mentoring the main graduate researcher on the project. How have the results been disseminated to communities of interest?Currently, two posters have been presented, one at the 2024 American Society of Microbiology meeting and another at the 2024 Gordon Research Confrence: Water. Additionally, a short overview slide was presented at the 2024 NIFA BRAG update. What do you plan to do during the next reporting period to accomplish the goals?In support of Objective 1, we will complete the selection of the in silico barcodes. These will be transformed into E coli and Baccilus subtilus (after recieving feedback at the 2024 ASM that this may be a beneficial strain to represent soil systems). In support of Objective 2, we will test these barcode transformed organisms in direct aerobic bioassays. In support of Objective 3 and 4, we will continue to establish the reactor system within the new dedicated space, with a goal of a full test run of the mock field and aquatic system by Q2 of 2025. The data will be curated and stored in a manner to support Objective 5.

Impacts
What was accomplished under these goals? During the establishment phase of this project, we have pursued a dual approach in achieving the computational and molecular biology methods. Within the computational realm, we established a computational pipeline in Python for unique watermark identification, the UnSaid pipeline. In using UnSaid, we have identified a list of over 800 testable minimal watermarks, achieving the goal of Objective 1 Task 1 (Identify GEM Watermarks). These watermarks will inform the first mutant screens. We additionally developed backup lists of various other lengths of watermarks to enable iterative screening if the first prioritized watermarks fail to be incorporated in a stable and non-interfering manner within either the host genome or the receiving plasmid. Within the molecular biology realm, we have established a competent Escherichia coli system with an inducible CRISPR-Cas expressing plasmid. This first test system demonstrated the uptake of plasmids, but also the introduction of a system that allows for direct genome modifications. We initially utilized a CRISPR-Cas 12 system to also develop a biosensor that can detect plasmid exchanges within communities, a critical element for monitoring the exchange of plasmids or uptake of naked DNA that is outline in Objective 2 Task 1 (Develop a standardized aerobic batch assay for quantifying the degradation of either naked DNA or GEMs in multiple media). We additionally are exploring direct transformation using classical approaches to introduce these watermarks as well. By pursuing a three-fold strategy early within the project, we are well-poised to achieve Objective 1 Task 2 (Insert Watermarks). This E. coli system was also grown in competition with a bacterial community from activated sludge as a demonstration of growth and recovery, an early trial of Objective 2 Task 1 and Task 2 (Determine the Community Impact). We additionally gained access to and began moving equipment to our dedicated test facility. This facility will house the model agricultural runoff network (Objective 4) and we are finalizing the design of these low-cost systems. This facility matches our needs well. We are constructing these model agricultural runoff system within triple secure access rooms, in separated environments. Within our design, we encase this space within polyethylene liner. When operating we will enter/exit with cleansuits. This ensures that contamination of the system is minimized and the sterility of those entering/existing. This allows us to pursue Objectives 1-4 in a parallel manner to ensure project success. In addition to the technical components, we onboarded a dedicated PhD student to the project, and anticipate transitioning another PhD student on the project in Spring 2025 for additional support. This graduate student already presented her initial work with the E. coli system at the American Society of Microbiology. The PI additionally presented a framework for genetically engineered microorganisms more broadly at the Gordon Research Conference: Water symposium. We additionally have identified an undergraduate researcher that will be continuing on with this project in Fall 2024.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: "Developing novel biosensors using CRISPR-Cas 12" Taylor Cason, Cresten Mansfeldt. Poster Presentation. 2024 American Society of Microbiology in Atlanata, Georgia June 19-23, 2024.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: "Anticipating the Future Impact of Synthetic Biology on Aquatic Sciences". Cresten Mansfeldt. Gordon Research Conference:Water, Holderness, New Hampshire. June 23-28, 2024