Our project, Lacto-Bexo, contributes to the iGEM community by identifying, characterizing, and validating a novel anti-adipogenic protein, hisF, from the probiotic Lactobacillus rhamnosus. We provide this discovery in the form of new, well-documented biological parts, a computational workflow for protein analysis, and a software-based method for quantifying experimental results.
We have submitted a collection of three new parts to the iGEM Registry. These parts provide the essential components for the expression of the hisF protein, which we have demonstrated to be a potent inhibitor of adipogenesis.
BBa_25VSVXX5 - hisF from _Lactobacillus rhamnosus* (Basic Part): This part contains the coding sequence for the Imidazole glycerol phosphate synthase subunit (hisF). Our research identified this exosomal protein as the active molecule responsible for inhibiting fat cell formation. The sequence was codon-optimized for expression in E. coli, making it a ready-to-use tool for teams interested in metabolic regulation, protein secretion, or developing novel therapeutics.
BBa_25AUDPFC - pET28b(+)-IPTG Inducible hisF (Composite Part): This composite part is the functional construct that validates our project’s core discovery. It consists of our hisF basic part (BBa_25VSVXX5) cloned into the pET28b+ expression vector. This design allows for high-level, IPTG-inducible expression of the hisF protein in _E. coli* strains like BL21(DE3). We experimentally confirmed that the protein produced from this construct inhibits lipid accumulation in adipocytes by up to 50%. Future iGEM teams can use this part to easily produce and purify hisF for their own research.
Our project contributes a valuable computational workflow for investigating protein structure and function, supported by a new dataset validating our wet-lab findings.
Comparative Structural Analysis of hisF Proteins: Using Colabfold, we predicted and compared the 3D structures of the hisF protein from the beneficial probiotic Lactobacillus rhamnosus against orthologs from common pathogens like Escherichia coli and Staphylococcus aureus and human AMPK. This analysis provides a computational framework for identifying unique structural features that may explain differential bioactivity.
Modeling of the hisF-AMPK Interaction: We used AlphaFold3 to model the direct interaction between the bacterial hisF protein and its human therapeutic target, the AMPK complex. Our in-silico measurements provide quantitative data supporting the unique nature of the L. rhamnosus protein. The interfacial Predicted TM-score (iPTM), which measures confidence in the predicted binding interface, was 0.28 for L. rhamnosus hisF. This was notably higher than the scores for hisF from other bacteria like E. coli (iPTM score of 0.15), offering a structural basis for the specific anti-adipogenic effect we observed in the lab.
To ensure robust and repeatable measurements, we are contributing a software-based tool for analyzing lipid accumulation.
ImageJ Macro for Lipid Droplet Analysis: We successfully employed an ImageJ macro to quantify lipid droplets from our Oil Red O stained microscope images.
This computational tool automates the measurement of key metrics such as Total Lipid Area (TLA), Average Droplet Size (ADS), and total Droplet Count (DC). We used this method to quantitatively confirm the dose-dependent inhibition effects of both L. rhamnosus exosomes and purified hisF protein.
Also, we classified the lipid droplet size into 3 categories 0-120, 120-240, >240um² and count the number of each categorized lipid droplets among negative control and conditioned treated to analyze the data qualitatively that both exosome and hisF not only inhibit the number of average lipid droplets but also diminishes the size of lipid droplet formation.
This protocol serves as a valuable contribution for future iGEM teams working on adipogenesis or similar cell morphology studies. We also documented our attempt to develop a new script for measuring redness intensity, contributing to the knowledge base for future software development in this area.
Our project’s success was built on a series of detailed and reproducible wet-lab protocols. We are contributing these complete methods to the iGEM community to facilitate future research in adipogenesis, bacterial culturing, and molecular biology.
1) 3T3-L1 Cell Culture and Adipogenesis: We provide a comprehensive guide for the culture and differentiation of 3T3-L1 preadipocytes. This includes protocols for:
Maintenance and Subculturing: Step-by-step instructions for routine culture, including media preparation and passaging techniques to maintain healthy, differentiation-competent cells.
Induction of Differentiation: A detailed timeline and recipes for the hormonal cocktail (MDI Initiation Medium containing Insulin, Dexamethasone, and IBMX) required to trigger the transformation of preadipocytes into mature, lipid-storing fat cells.
Oil Red O Staining: A validated protocol for fixing and staining differentiated cells to visualize and subsequently quantify lipid accumulation, the key endpoint for our assays.
2) Lactobacillus spp. Culturing and Processing: We detail the methods used to grow and process the bacterial strains for our experiments.
Bacterial Culture: A complete protocol for culturing Lactobacillus rhamnosus and other species, including the composition and preparation of MRS medium, inoculation, and incubation conditions.
Extracellular Vesicle (EV) Isolation: A step-by-step method for isolating bacterial exosomes from culture supernatant using sequential centrifugation and Amicon® centrifugal filters.
3) Molecular Biology Workflows: We offer end-to-end protocols for gene and protein expression analysis, which were used to elucidate the mechanism of action of hisF.
RNA Extraction and qPCR: A full workflow from cell lysis with TRIzol™ reagent to total RNA extraction, followed by cDNA synthesis and quantitative PCR (qPCR) analysis to measure the relative expression of target genes like Ppary, Cebpa, and Ampk.
Western Blotting: A complete guide to protein analysis, including protein extraction with RIPA buffer, quantification with a BCA assay, separation by SDS-PAGE, transfer to a membrane, and immunodetection of specific target proteins.
Cloning and Protein Expression: Detailed protocols for the entire cloning workflow, from PCR amplification and restriction digest to ligation, transformation, and final verification by Sanger sequencing. This is followed by a guide for expressing the recombinant hisF protein in the BL21(DE3) E. coli host system.
4) Bacterial Exosome Proteomics: We provide a full workflow for identifying the protein content of bacterial EVs, which was critical for discovering hisF.
Sample Preparation and Digestion: Proteins are reduced with DTT, alkylated with IAA, and digested into smaller peptides using trypsin. The resulting peptides are then desalted and purified using C18 solid-phase extraction.
LC-MS/MS Analysis: Purified peptides are separated by high-performance liquid chromatography (HPLC) and analyzed by tandem mass spectrometry to determine their mass and sequence fragmentation patterns.
Data Analysis: Raw spectral data is processed with a search algorithm (e.g., MaxQuant) to match experimental spectra against a protein sequence database, allowing for protein identification and label-free quantification.
Our team’s work contributes to future iGEM teams by integrating scientific rigor, human-centered design, and regulatory foresight into a unified model for responsible biotech innovation.
Through our Lacto-Bexo project, we developed and shared frameworks, methodologies, and open-access resources that can be adopted and scaled by other iGEM teams working at the intersection of synthetic biology, digital health, and human practices.
We created a Design Thinking–based workflow that helps iGEM teams systematically incorporate user empathy and ethical reflection into early-stage research design.
Conducted workshops modeled after Stanford d.school and IDEO methods.
Developed templates for Empathy Maps, Persona Profiles, and Journey Mapping, which can be reused by other teams to better understand user experiences.
Demonstrated how participatory design and stakeholder co-creation can guide experimental priorities and ethical decision-making in biotech projects.
Contribution: A transferable Human Practices toolkit that operationalizes user-centric bio-innovation.
We documented a step-by-step commercialization and policy-alignment process, connecting biological research with real-world impact.
Introduced a 5C Analysis Framework (Company, Customers, Competitors, Collaborators, Context) adapted for biotech startups.
Built a Business Model Canvas template that integrates scientific validation, regulatory readiness (GRAS, FDA), and sustainability goals.
Conducted market and policy headwind analysis (e.g., Korea’s R&D investment trends, global GLP-1 landscape) to show how biotech teams can align with national and institutional funding directions.
Contribution: A reproducible entrepreneurship module bridging lab research, compliance, and go-to-market strategy.
We emphasized inclusivity in both team collaboration and educational outreach.
Designed stigma-free narratives around obesity and weight management, ensuring communication materials avoided medicalized or exclusionary language.
Created bilingual (Korean–English) resources and visual communication templates that can help non-native English-speaking iGEM teams effectively present scientific work internationally.
Contribution: A replicable communication and inclusivity guideline for teams working in multicultural or health-sensitive domains.
We are releasing several open-access visual and analytical resources:
Editable versions of Market Analysis Charts, R&D Investment Maps, and User Behavior Diagrams (Python/matplotlib source files).
Design templates for MIRO-based co-creation sessions and Wiki infographics (Figma format).
Contribution: Freely reusable data visualization and co-design resources for iGEM teams working on complex interdisciplinary projects.
Our project not only offers a novel probiotic–exosome concept but also provides a framework for responsible biotech translation.
By combining wet lab validation, ethical engagement, regulatory analysis, and entrepreneurship education, we hope to empower future iGEM teams to bridge science and society more effectively.