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