Progress 08/15/24 to 08/14/25
Outputs
Target Audience:During this reporting period, the primary target audience reached was the broader scientific community, including researchers, faculty, graduate students, and industry professionals in the fields of synthetic biology, biomaterials, and biopolymer production. This was achieved through multiple presentations of project-related work titled Plodia interpunctella: A Novel Insect Platform for Rationally Designed Biopolymer Production. These outreach activities included: · Oral presentation at the Spring 2025 American Chemical Society (ACS) National Meeting, delivering the talk Plodia interpunctella: A Novel Insect Platform for Rationally Designed Biopolymer Production. · Poster and rapid oral presentation at the Fall 2024 Southeast Regional Society for Biomaterials (SFB) Meeting, presenting Plodia interpunctella Silk: A Pathway to Sustainable and Consistent Biomaterials. · Oral presentation at the University of Florida's Spring 2025 Biomaterials Day event, again highlighting Plodia interpunctella: A Novel Insect Platform for Rationally Designed Biopolymer Production. · Poster presentation at the 2025 Silk Proteins and the Transition to Biotechnologies Gordon Research Conference (GRC), where the work was shared on my behalf by another graduate student in the lab. In addition to conference outreach, I completed the 2024 Science Communications Course to further develop skills in effectively communicating complex research to diverse audiences. This period also involved direct engagement with undergraduate and pre-college students, fostering early-stage scientific training and interest in biomaterials research. Specifically: · Mentored four undergraduate researchers and one high school student, providing hands-on instruction in laboratory techniques directly supporting the project. · Supported mentee-led dissemination efforts, including four undergraduate poster presentations at University of Florida internal research symposia. · Guided one undergraduate mentee in preparing and delivering an oral presentation at the American Institute of Chemical Engineers (AIChE) 2024 Annual Meeting. I also advanced communication of project-related concepts and findings through peer-reviewed publications. During this reporting period, I had one review article accepted: Modifying Naturally Occurring, Nonmammalian-Sourced Biopolymers for Biomedical Applications. In addition, I submitted and resubmitted (with revisions) a research article titled Enhancing CRISPR Homology Directed Repair in IAL-PiD2 Insect Cells via Reagent Delivery Optimization and Cell Synchronization, which directly relates to improving the efficiency of genome engineering at the region of interest in Plodia interpunctella. Through these activities, the project reached a wide range of stakeholders, academic peers, early-career scientists, and students, expanding awareness of Plodia interpunctella as a novel biopolymer production platform and building capacity for future research in the field. Changes/Problems:Overall, the project remains on schedule, with no major changes to the approved research plan or data management plan. All objectives and milestones are expected to be completed within the original project timeline. One temporary challenge occurred during work on Objective 1 (quantifying silk production and ease of collection). Our primary environmental control incubator experienced a humidity regulation failure, which limited our ability to collect a complete dataset across all rearing box designs under optimal conditions. While this delayed full completion of Objective 1, the downtime was used productively to advance Objective 2 (optimizing CRISPR/Cas9-mediated gene editing in Plodia, and creation of a transgenic line of Plodia), allowing us to advance Objective 2 ahead of schedule. The incubator has since been repaired, and data collection for Objective 1 is resuming. Looking ahead, a potential hurdle is the possibility of altered fecundity in genetically modified Plodia strains. Reduced reproductive output could affect long-term scalability and strain maintenance. This will be monitored closely in the next reporting period, with dedicated experiments to quantify any changes in fecundity and assess their implications for production efficiency. No significant deviations in research scope, schedule, or goals have occurred, and no changes in protocols for the use or care of animals, human subjects, or biohazards were required during this reporting period.? What opportunities for training and professional development has the project provided?Training Activities This project provided extensive hands-on training opportunities for undergraduate and pre-college researchers. Four undergraduate students and one high school student worked directly on the project under my mentorship. Training focused on developing technical proficiency in molecular biology, microbiology, and biomaterials characterization techniques, including CRISPR/Cas9 genome editing strategies, insect rearing and husbandry, microscopy, DNA, RNA and protein extraction, and silk fiber analysis. Mentees also gained experience in experimental design, data collection, data analysis, and scientific record keeping. Beyond laboratory skills, I supported their professional growth by guiding the preparation of abstracts, posters, and oral presentations. As a result, mentees presented their work at multiple venues: four posters at internal University of Florida research symposia and one oral presentation at the American Institute of Chemical Engineers (AIChE) 2024 Annual Meeting. These experiences provided them with valuable networking opportunities, exposure to the broader scientific community, and the confidence to communicate their work effectively to technical and non-technical audiences. Professional Development Activities Through this project, I expanded my own professional expertise in both scientific communication and dissemination. I presented project results through multiple invited and competitive presentations, including an oral presentation at the Spring 2025 American Chemical Society National Meeting, an oral presentation and poster at the Fall 2024 Southeast Regional Society for Biomaterials Meeting, an oral presentation at the University of Florida's 2025 Biomaterials Day, and a poster presentation at the 2025 Silk Proteins and the Transition to Biotechnologies Gordon Research Conference (presented on my behalf by a colleague). These activities strengthened my skills in delivering research findings to diverse scientific audiences and facilitated collaborations across disciplines. How have the results been disseminated to communities of interest?Results from this project have been actively shared with national, regional, and institutional research audiences to ensure that findings reach scientists, engineers, and industry professionals interested in biomaterials, synthetic biology, and recombinant protein production. This included: · Oral presentations at the Spring 2025 American Chemical Society National Meeting and the University of Florida's 2025 Biomaterials Day, presenting Plodia interpunctella: A Novel Insect Platform for Rationally Designed Biopolymer Production. · Poster and rapid oral presentation at the Fall 2024 Southeast Regional Society for Biomaterials Meeting (Plodia interpunctella Silk: A Pathway to Sustainable and Consistent Biomaterials). · Poster presentation at the 2025 Silk Proteins and the Transition to Biotechnologies Gordon Research Conference, delivered by a lab colleague on my behalf, to reach an international audience of silk biologists and biomaterials researchers. In addition, project-related manuscripts are being used to disseminate results through peer-reviewed publications. A research paper on CRISPR/Cas9 optimization in Plodia PiD2 cells, Enhancing CRISPR Homology Directed Repair in IAL-PiD2 Insect Cells via Reagent Delivery Optimization and Cell Synchronization, has been submitted, reviewed, and resubmitted with revisions to the Biochemical Engineering Journal. A review article, Modifying Naturally Occurring, Nonmammalian-Sourced Biopolymers for Biomedical Applications, has been accepted, and a third manuscript describing fluorescent recombinant silk production is in preparation. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will build upon the progress made this year to advance both our silk production optimization and genetic engineering goals. 1. Quantifying Silk Production and Ease of Collection o Conduct controlled experiments to fully quantify silk yield across rearing box designs, ensuring environmental conditions (including humidity) remain stable. o Assess the ease of silk collection from each design and generate standardized collection protocols. o Prepare and submit a manuscript reporting these findings, with undergraduate mentees contributing to both experimental work and data analysis. This will also provide them with opportunities to develop scientific writing skills and present their results at internal and external conferences. 2. Publication Goals o Finalize and publish the CRISPR/Cas9 optimization study (Enhancing CRISPR Homology Directed Repair in IAL-PiD2 Insect Cells via Reagent Delivery Optimization and Cell Synchronization) in the Biochemical Engineering Journal. o Complete the manuscript describing the mNeonGreen-fusion silk, highlighting its utility as a visible marker for recombinant silk production. 3. Advancing Genome Engineering Capabilities o Implement a newly designed two-step CRISPR recombinase-mediated cassette exchange (RMCE) strategy to allow repeated use of the same genomic site for introducing larger, more complex sequences. o Design and construct new DNA cassettes for testing RMCE efficiency and versatility in Plodia. o Evaluate the success of sequence integration and expression in modified lines. 4. Characterizing Recombinant Silk Properties o Assess the mechanical and physical properties of recombinant silk fibers produced using the new constructs. o Utilize bio-atomic force microscopy (bio-AFM) and rheology to measure fiber strength and elasticity By the end of the next reporting period, these activities will produce a comprehensive dataset on both native and recombinant silk production in Plodia, generate at least two peer-reviewed publications, and expand our genetic engineering platform to support more advanced recombinant silk designs. This will directly address our goals of establishing Plodia interpunctella as a reliable U.S.-based platform for silk production and biomanufacturing.
Impacts
What was accomplished under these goals?
Silk is a strong, lightweight, and biodegradable material that has been valued for thousands of years in textiles, cosmetics, and medicine. It offers a sustainable alternative to petroleum-based plastics, yet producing silk in the United States has remained challenging. Traditional silk farming depends on outdoor cultivation of the domesticated silkworm (Bombyx mori), which is labor-intensive, costly in the U.S., and vulnerable to climate change. As a result, the U.S. has missed opportunities in the expanding global silk market. Plodia interpunctella (Plodia), the Indian meal moth, offers a promising alternative. It can be reared entirely indoors under controlled conditions, enabling consistent year-round production and eliminating many environmental risks. Importantly, Plodia is not only capable of producing its native silk but can also be genetically engineered to produce recombinant silks fused with valuable proteins for biomedical, industrial, and specialty textile applications. Over the past year, our project made measurable progress toward unlocking Plodia's potential. We designed and tested four 3D-printed inserts to increase rearing box surface area, from the original 1,032 cm² to as much as 1,920 cm², allowing more space for larvae to spin silk. Early tests, despite temporary humidity issues, showed promising gains: one design more than doubled silk yield (187.15 mg vs. 78.62 mg in controls). On the genetic engineering side, we developed a panel of sgRNAs targeting the N-terminal, middle, and C-terminal regions of the Plodia fibroin heavy chain gene, providing flexibility to modify the protein's length in either direction. Our main focus in year one was on C-terminal targeting, as this region offered the highest likelihood of successful integration and the ability to extend the protein by adding native repeat sequences. We then optimized CRISPR/Cas9 editing in the Plodia PiD2 cell line by adjusting homology arm length, Cas9-to-guide RNA ratios, and DNA template amounts, achieving an 11% precise integration rate. These improvements were validated by inserting a fluorescent reporter gene and creating transgenic Plodia that produce silk fused to mNeonGreen, allowing easy visual tracking of modified silk production. Our dissemination of these findings is already underway. A research manuscript detailing the CRISPR/Cas9 optimization in Plodia PiD2 cells, Enhancing CRISPR Homology Directed Repair in IAL-PiD2 Insect Cells via Reagent Delivery Optimization and Cell Synchronization, has been submitted, peer-reviewed, and resubmitted with revisions. These outcomes mark significant progress in both knowledge and capability. We now have a tested approach to increase native silk yield indoors and a functional genetic toolkit for producing recombinant silk with specialized properties. This dual capability means Plodia could supply high-quality silk for conventional textiles while also enabling entirely new products, such as silk-based medical implants, drug delivery materials, or high-performance fibers. At scale, these methods could supply thousands of kilograms of silk annually, strengthen domestic manufacturing, create new biotech jobs, and reduce U.S. dependence on imported silk, bringing economic, environmental, and technological benefits to multiple industries.
Publications
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