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

Outputs
Target Audience:We are targeting several audiences for sharing our materials and methods for the plant-model system and the knowledge we gain into bioinoculant developments that ensure safe use in field settings. Our target audiences include: 1. Biotechnology companies: Our project will serve biotech researchers in agriculture by providing a framework for the evaluation of genetic containment strategies. Our genetically tractable and traceable Rhizobium species and plant-soil system will provide the means to evaluate and compare evasion risk among containment strategies. These tools will improve confidence in the deployment of future biotechnologies in plants and soil systems. In 2025, PD Wilhelm remains a member of the Coordinating Committee of the Phytobiomes Alliance and has maintained a relationship with researchers at Ginkgo Bioworks to explore the use of our model system. PD Wilhelm has developed a new relationship with the CEO of Lilac Agricture, Dr. Barney Geddes, to begin testing his isolate libraries in our model plant-soil system to identify characteristics of elite strains. Our team values inputs from industry partners, as their adoption of our approach is critical to its success. Graduate student Machado received strong positive feedback from industry partners from his presentation on "Evaluating Bioinoculant Efficacy through a Dynamic Plant-Soil-Microorganism Interaction Model" at the Biostimulants World Congress in November, 2024. Graduate student Machado also presented at the Phytobiomes Alliance meeting, describing work completed for the BRAG project (completed in 2023), entitled: "Factors Influencing the Use of PNA Clamps for Surveys of Endophyte Communities" in November, 2024. 2. Agronomists and Growers: Biotechnologies are low cost and scalable for agricultural applications. Currently in the USA and globally, there is a surge of interest in their efficacy for biocontrol, biofertilization etc. PD Wilhelm has presented at the Phytobiomes Alliance conference (Nov. 2024) and, via connections made there, PD Wilhelm is leading a perspectives paper on bioinoculant technologies entitled: "Precision agriculture meets phytobiome management: Challenges and opportunities for innovation in broad-acre agricultural systems," which has brought expertise from economists, agronomists, and industry partners. PD Wilhelm continued to engage with growers and agronomists to learn about their needs and uses of biotechnologies. PD Wilhelm has presented at 2 grower meetings in sponsored by AgSpectrum (January, 2025) about developments in biotechnologies and the need for biocontainment strategies. Graduate student Machado presented an overview of his BRAG research, entitled "GEM in Agricultural Systems," at the Higher Education Challenge: Communicating Agriculture Beyond Higher Education Conference in Miami in October, 2024. 3. Classroom and laboratory instruction: The expertise we develop in our project will contribute to foundational growth in synthetic biology at Purdue. Co-PD Green is the supervisor of Purdue's International Genetically Engineered Machine (iGEM) team and, in 2024-25, graduate students Gonzalez and Cook have been mentoring two iGEM teams. These teams have not yet competed, as the program was reinitiated by Green in 2024. Preparations included formulating team structure, funding sources, and the setup of a 'maker space' designated for iGEM activities. One iGEM team is working on Rhizobium etli as a direct extension of the BRAG project. PD Wilhelm and Co-PD Green are board members of the Purdue Applied Microbiome Sciences and are steering the group to develop graduate training workshops in synthetic biology. While this ambition will not have an immediate impact beyond undergraduate and graduate students on our campus, the goal is to build expertise that serves early career scientists in the Midwest. This ambition is manifested in the growth of the Purdue Microbiome Symposium into the Midwest Microbiome Symposium in the past 4 years, which has been led by PD Wilhelm and Co-PD Green who sit on the organizing committee. Changes/Problems:We have not yet encountered any problems that have required us to switch course. We are approaching a decision point regarding the feasibility of a cold-sensitive promoter for the kill switch. This was identified as a priority given the fact a plant will be stressed at higher temperatures where the currently validated promoters function. It took us 1 year to recruit our SynBio team, which has delayed progress on the GEM engineering, but students Cook and Gonzalez are now situated and making more efficient progress. Student Machado brings 3 years of scholarship funding, so we have run-way to complete all proposed work. It is just taking some time to synch up. With awards and budgeted funds, we have the capacity to support both MS for two years and our PhD candidate for a potential 5 years. What opportunities for training and professional development has the project provided?PhD student, Grace Cook, participated in the prestigious Cold Spring Harbor Laboratory Synthetic Biology 2-week summer course (Summer 2024) supported by funds from this grant. All students (Cook, Gonzalez, and Machado) received training on Oxford Nanopore sequencing performed on the genomes of Rhizobium isolates and for plasmid libraries containing kill switch constructs and DNA barcodes. The sequencing runs demonstrated that full-length circular plasmids could be recovered with sufficient coverage to confirm construct integrity and to identify barcodes and potential escape variants. These sequencing tools complement the development of quantitative assays, which include qPCR protocols designed to measure survival ratios using ReMark. How have the results been disseminated to communities of interest?Students have participated in two dissemination / outreach events: 1. StepN2STEM Event, February 2025, Purdue University, West Lafayette, IN. This involved setting up a table to teach about synthetic biology and its applications to topics in agriculture and health. The table included hands-on activity utilizing electronics with snap connections that light up an LED to explain the activation of a gene. 2. Purdue iGEM - 2024-25 Teams. Students Cook and Gonzalez are both serving as Purdue iGEM Graduate Student Advisors. In this capacity, they each mentor a team focused on a bioengineering task. Both teams are developing ideas related to this BRAG project. One related to ReMARK and the other to cold temperature adaptations (for regulating the kill switch). What do you plan to do during the next reporting period to accomplish the goals?In Year 3, our team needs to focus on finalizing and validating the cold temperature-sensitive Rhizobium strain. Cook is pursuing two strategies that will be tested within the first half of Y3. If these fail, we will engage in our pitfall planning to work with established temperature regulators at higher temperatures (30 °C). Gonzalez will be finalizing the kill switch circuits and working with a postdoc in Dr. Barney Geddes's lab to facilitate the transformation and integration into R. etli. Student Machado will publish a description of his synthetic soil and, while waiting for the SynBio team to generate the GEM, he will be conducting experiments with plasmidID labeled rhizobia testing the influence or rhizosphere competition on survival. He will complete the development and testing of qPCR primers targeting our isolate.

Impacts
What was accomplished under these goals? Objective 1. Engineering Rhizobium and Developing Molecular Tools to Assess Evasion Risk The purpose of Objective 1 is to genetically engineer Rhizobium and develop quantitative tools that assess evasion risk in the plant and soil compartments of the Phaseolus-Rhizobium model system. We made progress on both sub-objectives: (1.1) engineering of R. etli, and (1.2) development of molecular methods to quantify kill switch efficacy. Objective 1.1: Engineering of R. etli Progress this year established a foundation for kill-switch engineering. Students constructed and characterized several toxin-based kill switches using modular 3G cloning. These included a CcdB-CcdA toxin-antitoxin system, the phage-derived polymerase inhibitor Gp2, and the φX174 lysis protein. The kill switch cassettes were placed under inducible LacI/pLac control and cloned into both E. coli vectors and a broad-host-range conjugative shuttle vector (pBBR1_DVK_AE). When tested in E. coli BL21 DE3, the constructs displayed induction-dependent toxicity, with the φX174 lysis protein producing the most pronounced killing response. Efforts also focused on establishing transformation protocols for rhizobia. An adapted electroporation protocol, originally designed for Agrobacterium, enabled plasmid introduction into Rhizobium tropici CIAT 9030, verified through constitutive expression of fluorescent reporters (sfGFP and mScarlet). In parallel, conjugation protocols using E. coli S17 λpir were optimized, providing an alternative DNA transfer method. Despite these advances, kill switch constructs introduced into R. tropici did not produce the expected toxicity. The lack of IPTG uptake due to absence of LacY permease, toxin incompatibility with rhizobial physiology, and promoter/codon usage issues likely contributed. To address these, planned strategies include testing Rhizobium-specific phage toxins, identifying alternative promoters not dependent on IPTG, and exploring genomic integration systems such as CRISPR-associated transposons. Objective 1.2: Development of Molecular Methods We made progress on designing ReMark, though the task is incomplete. Several approaches were discussed, focusing on designing a synthetic RNA marker with improved stability--short 5′ UTRs, increased GC content, and unique primer binding sites. Strategies to overcome RNA instability in soil and plant environments included multiple hairpins, "dummy RNAs" with enhanced folding, and RNA condensates or nanostructures to resist degradation. Parallel experiments tested spiked bacterial RNA recovery from synthetic soil. DNA was consistently detected, but RNA recovery was unreliable, underscoring the need for stabilization strategies and improved protocols. No single design is finalized, but exploration clarified barriers and established a menu of strategies. The next stage will select promising designs for synthesis and testing, aiming to produce a robust ReMark system by Q3 of Y3. Objective 2. Characterization of Evasion Risk in Plant-Soil Compartments The purpose of Objective 2 was to characterize the evasion risk of ReGEM in Phaseolus-Rhizobium plant-soil compartments. This involved validating molecular methods for detection in complex matrices and evaluating colonization dynamics in controlled plant experiments. Synthetic Soil Development To ensure reproducibility and control, a synthetic Alfisol-based soil matrix was developed and optimized. The formulation replicated key mineralogical and textural traits of natural Alfisols, incorporating quartz sand and silt, calcium bentonite, muscovite, and kaolinite. Organic matter components were modeled with polyglutamic acid, cellulose, lignin, yeast biomass, and xanthan gum to promote aggregation. Soil was sterilized through three autoclave cycles, with sterility validated by absence of microbial growth. Refinements improved performance: substitution of calcium for sodium bentonite to lower sodium and sodium adsorption ratio, exclusion of hemicellulose for cost efficiency, C:N ratio increase from 7.4 to 15, and xanthan gum addition for aggregate stability. Controlled Plant Experiments Using this soil platform, a potted plant experiment was conducted with four elite rhizobial strains (R. etli CFN42, R. tropici CIAT889, R. phaseoli USDA2668, R. leguminosarum bv. phaseoli USDA2671). Seedlings were inoculated at planting with 1 × 10? cells/mL, and plants were grown for sixty days. The synthetic soil supported healthy plant growth, and inoculated rhizobia successfully formed nodules. Harvested tissues (leaves, roots, nodules, pods) are being processed for DNA extraction to determine which strains most effectively colonize each compartment and provide insight into compartment-specific survival and persistence. Development of Molecular Detection Tools To quantify ReGEM in plant and soil matrices, RNA recovery protocols were evaluated using spiked soil samples. Comparisons between commercial and custom methods showed phenol-chloroform extraction outperformed the ZymoBIOMICS DNA/RNA kit, especially at low bacterial concentrations. While the kit failed at 2.5 × 10? cells, the phenol-chloroform method recovered measurable RNA. Plans are underway to expand testing with QIAGEN's PowerSoil RNA kit and plant RNA kits to improve plant-compartment compatibility. These optimizations are essential for developing reliable assays to track engineered rhizobia in complex matrices. Objective 3. Environmental and Ecological Drivers of Evasion Risk The aim of Objective 3 is to identify environmental and ecological factors shaping variation in evasion risk using controlled Phaseolus-Rhizobium experiments. Considerable progress was made in developing experimental design, though implementation has not begun. The team finalized experiments varying abiotic stressors, rhizosphere competition, and free-living survival. Each study targets ~50 units, balancing ecological relevance with tractability. Across experiments, pH and soil texture serve as master variables, while treatments manipulate soil moisture, salinity, nutrients, microbial competition, and organic matter. Coordination with collaborators secured resources and microbial materials. Agreements to share mutant libraries and synthetic soil components ensure readiness. The iterative design process refined treatments for interpretability and feasibility. Although execution has not started, the project is well-positioned to proceed. Experimental designs are complete, materials secured, and clear hypotheses articulated. This preparation ensures forthcoming experiments will directly address how abiotic and biotic factors influence ReGEM persistence and evasion risk in plant-soil environments.

Publications

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

Outputs
Target Audience:We are targeting several audiences for sharing our materials and methods for the plant-model system (ex. bacterial strains) and the knowledge we gain into the escape risk of genetic containment strategies in natural settings. Our target audiences include: 1. Biotechnology companies: Our project will serve biotech researchers in agriculture by providing a framework for the evaluation of genetic containment strategies. Our genetically tractable and traceable Rhizobium species and plant-soil system will provide the means to evaluate and compare evasion risk among containment strategies. These tools will improve confidence in the deployment of future biotechnologies in plants and soil systems. PD Wilhelm is a member of the Coordinating Committee of the Phytobiomes Alliance and has been familiarizing industrial partners with the aims of our project, including Syngenta, PivotBio, Earth Microbial, and Ginkgo Bioworks. We have established a relationship with researchers at Ginkgo Bioworks to explore the use of our model system, as well as share resources and expertise. 2. Agronomists and Growers: Biotechnologies are low cost and scalable for agricultural applications. Currently in the USA and globally, there is a surge of interest in their efficacy for biocontrol, biofertilization etc. We will engage with growers and agronomist to learn about their needs and to share what we learn about developments in biocontainment strategies. In the USA, we will reach these audiences at the national level at the annual Tri-Societies meeting (Agronomy, Crop, and Soil Science Societies of America) and via PD Wilhelm's annual extension outreach events in Indiana, including the Indiana Certified Crop Advisors Conference, and the Indiana Soil Health and Sustainability Course. We also plan to engage audiences outside of the USA, particularly in Latin America, where beans are a staple crop. The PhD student leading the work, Túlio Machado, is a Brazilian national who has developed collaborations with researchers in Mexico (Dr. David Romero), Brazil (Dr. Mariangela Hungria da Cunha), and Spain (Dr. Beatriz Jorrin Rubio). 3. Classroom and laboratory instruction: The expertise we develop in our project will contribute to foundational growth in synthetic biology at Purdue. Co-PD Green is the supervisor of Purdue's International Genetically Engineered Machine (iGEM) team and both incoming MS students will be closely involved in mentoring the team. PD Wilhelm and Co-PD Green are board members of the Purdue Applied Microbiome Sciences and are steering the group to develop graduate training workshops in synthetic biology. While this ambition will not have an immediate impact beyond undergraduate and graduate students on our campus, the goal is to build expertise that serves early career scientists in the Midwest. This ambition is manifested in the growth of the Purdue Microbiome Symposium into the Midwest Microbiome Symposium in the past 3 years, which has been led by PD Wilhelm and Co-PD Green who sit on the organizing committee. Changes/Problems:We have not yet encountered any problems that have altered our plans. We have changed our initial timeline by approximately 6 - 8 months due to delays in our recruitment of a MS student to undertake R. etli engineering in Objective 1. Students with training in synthetic biology are in high demand. We sought to entice talented students with entrance scholarships and conducted an extensive search for suitably trained students. From these efforts, we managed to recruit two MS students (Grace Cook and Luis González) to work on Objective 1 and were successful in obtaining a one-year fellowship for Luis González. Both incoming MS students have extensive backgrounds in bioengineering. Túlio Machado, the PhD responsible for Objectives 2 and 3, was also awarded a three-year fellowship. With awards and budgeted funds, we have the capacity to support both MS for two years and our PhD candidate for a potential 5 years. What opportunities for training and professional development has the project provided?Incoming MS student, Grace Cook, was admitted to the prestigious Cold Spring Harbor Laboratory Synthetic Biology 2-week summer course (Summer 2024). Funds from this grant are supporting this training opportunity. How have the results been disseminated to communities of interest?The current PhD student has given seminars on two topics: Machado, Túlio. The use of PNA clamps for surveys of plant bacterial diversity. Agronomy Graduate Student Organization Seminar Series. Purdue University. April 29th, 2024. Machado, Túlio. Kill Switches for GEM in Agriculture. Purdue Applied Microbiome Sciences. Purdue University. December 1st, 2024. What do you plan to do during the next reporting period to accomplish the goals?In Year 2, our team will make significant advances in the engineering of the cold temperature-sensitive Rhizobium strain. MS students Grace Cook and Luis Gonzalez will be trained to engineer Rhizobium. MS student, Grace Cook, will be responsible for engineering and testing the ccdB-ccdA kill switch circuit in Rhizobium (currently in E. coli) and for developing a Cas9-mediated kill switch circuit. MS student, Luis Gonzalez, will be responsible for tuning a library of temperature sensitive promoters in Rhizobium and using them to regulate the kill switches. PhD student, Tulio Machado, will finalize the methodological setup for version 1 of the model plant-soil system. He will complete the development and testing of qPCR primers targeting our isolate. He will have performed in soil and in plant competition assays with our GEM and close relatives of Rhizobium.

Impacts
What was accomplished under these goals? We have accomplished several tasks in each of our Objective areas: Objective 1. To establish standardized procedures and quantitative methods for assessing evasion risk in a Phaseolus-Rhizobium model system. We identified and finalized several design elements of our model system to ensure it can be readily adopted and widely used for testing and risk assessment. To this end, we performed two major surveys to select the optimal cultivars of Phaseolus (for growth in a containment facility) and strains of Rhizobium (for colonization performance and ease of transformation) as described in Activity 2. We are currently running our first potted plant experiment testing the colonization of P. vulgaris Benton by our three candidate Rhizobium: the two engineered strains (R. etli CN42 and R. leguminosarum 3841) and one native strain R. tropici, which is known to be among most effective nodulators of P. vulgaris. The experiment will test whether there are differences in the characteristics of colonization by our different Rhizobium strains, and to generate samples for testing our quantification methods. We have made progress on two quantification methods for calculating the survivor ratio (cells that survive under non-permissive conditions): (i) qPCR targeting our engineering bacteria ('reMarker') and (ii) the use of fluorescent protein markers (GFP) and flow cytometry. We have designed and are currently testing qPCR primer sets for the family Rhizobiaceae (targeting the recA gene) and targeting the GFP gene (a marker of our engineered strains). We have begun designing our unique mRNA construct ('reMarker') to optimize detection of surviving cells by strengthening expression and the RNA sequence that has the greatest stability (i.e. the lowest minimum-free-energy change). Objective 2: To compare the efficacy of programmed death and the frequency and type of escape mutants in plant (nodule, root, leaf, and seed) and soil compartments (rhizosphere, soil leachate, and bulk soil). In Year 1, our main goal for Objective 2 was to engineer inducible ccdB expression. To that end, we accomplished two tasks: (1) to establish protocols for engineering kill switches in the E. coli Marionette strain system and (2) to acquire and develop the genetic components and workflow to transform R. etli strains. Task 1: The Marionette strain contains a 12.6 kb insert that expresses 12 small-molecule inducible transcription factors, or "sensors," and has been integrated into the glvC locus of three E. coli strains (MG1655, DH10B, and BL21). This system is valuable for optimizing gene expression dynamics in synthetic bacteria. We modified the plasmid pSC101 by introducing the ccdB gene, which produces a toxin that inhibits DNA gyrase activity that causes cell death. Specifically, we cloned the ccdB gene under the control of the pLac promoter into the MG1655 strain of the Marionette using single-day construction of 3G assembly to create a modular plasmid for heat shock transformation. We confirmed the efficacy of the kill switch by performing a growth inhibition assay using our new E. coli marionette chassis with no IPTG or 1mM of IPTG. We measured and plotted the RFP fluorescence normalized by OD 600 over a 24-hour incubation period. The IPTG-inducible ccdB circuit demonstrated the ability to constrain the bacterial culture population to a low density when introduced at the start and at a subsequent stage of growth. Gyrase inhibitors inhibit cell growth, resulting in residual cell remnants in the solution. In Year 2, we will be optimizing the cell death components in E. coli, including lysis and Cas-based kill switches. Task 2: In Year 1, We have obtained genetically competent Rhizobium strains, which will be transformed with our kill switch circuit. We have acquired Rhizobium etli CFN42 with integrase that catalyzes the direct recombination into the chromosome, creating a stable construct. This will serve our aim of integrating our kill switch circuit into the host genome, to improve the stability of the chassis being used in our model plant-soil system. To hedge our bets, we also have a strain of R. leguminosarum 3841 harboring a mini-Tn7-Gm cassette with GFP integrated in the chromosome with gentamycin as an antibiotic marker. Over the next three months (July through September 2024), the Green Lab will develop a library of genetic components for engineering responsive kill switches in R. etli. We will identify and order (via IDT gBlocks) sequences for inducible quorum sensing receptors and protomers, a library of ribosome binding sequences, and toxin output genes (e.g., optimizing ccdB or expressing yacG in Rhizobium, viral lytic broad-range Rhizobium genes). The engineering of our chassis should be robust for our model system to be useful to our intended users. In Year 2, we will also optimize the protocol for transforming Rhizobium strains to enhance the uptake of synthetic DNA using electrocompetent, chemically competent, and a novel protocol the Green Lab is establishing for DNA nanopore-competent transformation. We aim to demonstrate robust and efficient methods for transferring synthetic DNA plasmids across R. etli membranes for engineering novel circuits in the GEM chassis. Objective 3: To assess the influence of environmental (resources and stress) and ecological drivers (competition) on evasion risk. This objective will be focused on in Year 3, but we have begun preparation for the experiments. We demonstrated that the PNA clamp can be used to determine diversity and composition of the endophyte and rhizosphere microbiome of P. vulgaris, which is necessary to study the ecological drivers of evasion risk. We have also started a collection of Rhizobium and closely related members of Rhizobiaceae for use in controlled experiments to test the influence of competition on the survival of our target GEMs in non-permissive conditions.

Publications