Fleur Design
Fleur is a live biotherapeutic product (LBP): a genetically engineered probiotic designed to synthesize and secrete native, wild‑type human GLP‑1 directly within the gastrointestinal (GI) tract. By leveraging synthetic biology to transform a safe probiotic into a localized drug vehicle, Fleur aims to mimic the physiological pattern of GLP‑1 action (pulsatile, meal‑responsive, and locally concentrated) thereby preserving therapeutic efficacy while minimizing the systemic exposure that drives adverse effects. This approach represents a paradigm shift from conventional pharmacology to a new class of engineered, living medicines.
Probiotics Design
Fleur's core hypothesis is that by delivering wild‑type GLP‑1 locally within the gut, it is possible to achieve therapeutic benefit while avoiding the consequences of systemic overexposure. This is accomplished by engineering a probiotic chassis to function as a controllable, in‑situ biomanufacturing system.
Fleur Design
Chassis Selection
The choice of the bacterial host is paramount. We selected Lactobacillus plantarum and Lactobacillus acidophilus, lactic acid bacteria with several key advantages. Both have a long history of safe human consumption in fermented foods and are designated as Generally Recognized As Safe (GRAS) by the U.S. FDA [1,2]. Both possess natural tolerance to the low pH of the stomach and the bile salts of the small intestine [3]. Finally, a well‑established suite of genetic tools, protocols, and usage exists for the two species [4]. Reference: Optibac Probiotics.
Optibac Probiotics
Theoretical Genetic Construct Design
The therapeutic function is encoded in a custom‑designed genetic expression cassette, stably integrated into the chromosome. Key components include a promoter (constitutive initially; inducible in advanced designs to couple expression to meals), a codon‑optimized GLP‑1 gene (for Lactobacillus translation efficiency), and a secretion signal peptide (e.g., Usp45 from Lactococcus lactis) that directs export of GLP‑1 to the gut lumen [5]. Chromosomal integration promotes genetic stability and reduces mobilization risk compared to plasmids [6].
Once consumed, engineered L. plantarum reaches the small intestine and secretes human GLP‑1, establishing a high local concentration at its primary site of action. Circulating GLP‑1 is rapidly degraded by DPP‑4, re‑creating a physiological gradient—high in the gut, low in the blood—thereby minimizing systemic side effects.
Fleur Design
Engineered Plasmid Design
Plasmid Design
| Component | Part | Flanking sequences |
|---|---|---|
| Vector | JUMP BBa_J428326 | 5' GGAG / 3' AATG |
| CDC | CRISPR‑CaS BBa_J428055 | 5' AATG / 3' GCTT |
| Terminator | E. coli Protein Expression BBa_J435361 | 5' GCTT / 3' CGCT |
| Right Connector | Open Yeast (species agnostic) BBa_J435238 | 5' CGCT / 3' AGAC |
| Left Connector | Open Yeast (species agnostic) BBa_J435232 | 5' ATGA / 3' GGAG |
Experimental Parts
Source: iGEM Distribution Kit
| Plate | Well | Name | Description |
|---|---|---|---|
| 1 | I1 | amajLime | E. coli Protein Expression – fluorescent protein gene |
| 1 | G1 | tsPurple | E. coli Protein Expression – fluorescent protein gene |
| 1 | O1 | aeBlue | E. coli Protein Expression – fluorescent protein gene |
| 1 | C3 | eYFP | E. coli Protein Expression – fluorescent protein gene |
| 1 | M1 | asPink | E. coli Protein Expression – fluorescent protein gene |
| 1 | A15 | 3_Colors_ins_BBa_J06504 | E. coli Protein Expression – fluorescent protein gene |
| 1 | K1 | gfasPurple | E. coli Protein Expression – fluorescent protein gene |
| 1 | I15 | GFP (green) | E. coli Protein Expression – fluorescent protein gene |
| 1 | E3 | EiraCFP (cyan) | E. coli Protein Expression – fluorescent protein gene |
| 1 | M3 | mCerulean | E. coli Protein Expression – fluorescent protein gene |
| 2 | G1 | cjBlue | E. coli Protein Expression – fluorescent protein gene |
| 2 | C7 | amilRFP | E. coli Protein Expression – fluorescent protein gene |
Challenges and Solutions
Developing a living medicine presents unique challenges not found in conventional drug development.
Challenge 1: Dosage Control
Challenge: How can one control the dose of a drug that is produced by a self-replicating organism? The population dynamics of the engineered probiotic in the gut are complex.
Solution – Transient Colonization & Regular Dosing: Most probiotics, including L. plantarum, are transient colonizers cleared within days to weeks. A stable therapeutic effect relies on regular consumption (e.g., daily), which creates a predictable steady‑state population. Dose is controlled by viable cell concentration and dose frequency.
Solution – Engineered Biocontainment: Advanced containment circuits (e.g., engineered auxotrophy) constrain bacterial proliferation by requiring a nutrient supplied only in the formulation, absent in the gut [7].
Challenge 2: Genetic Stability & HGT
Challenge: Genetic Stability and Horizontal Gene Transfer (HGT): A primary safety concern is the stability of the engineered gene cassette and the potential for it to be transferred to other resident gut microbes.
Solution: Chromosomal integration at safe‑harbor loci reduces horizontal transfer risk and increases stability versus plasmids [6]. Integration sites are chosen away from mobile genetic elements to minimize HGT potential.
Challenge 3: Host–Microbe Interaction
Challenge: Host-Microbe Interaction and Immune Response: Introducing a genetically modified organism could potentially alter its interaction with the host immune system.
Solution: Lactobacilli generally trigger tolerogenic/anti‑inflammatory responses [8], but preclinical validation (co‑culture, animal immunology) is required to confirm that human GLP‑1 expression does not provoke adverse inflammatory or allergic responses.
Computational Engineering
We prioritize GLP‑1 amino‑acid positions amenable to mutation using multiple sequence alignment (MSA) and Shannon entropy to improve stability and receptor binding while preserving structure.
Identifying Target Sites for Mutation
- Low entropy (< 1.0): Highly conserved and critical – avoid modifying.
- High entropy (> 2.0): Variable and tolerant – preferred engineering targets.
Our initial focus is on the four most abundant amino acids in GLP‑1: Leucine, Serine, Glycine, and Alanine.
Clinical Application and Future Vision
Fleur will follow the LBP regulatory pathway from preclinical safety/efficacy through phased clinical trials. Beyond GLP‑1 for diabetes and obesity, the platform can deliver other proteins in the gut: IL‑10 for IBD [9], PAL for PKU [10], or anti‑infective peptides targeting pathogens such as C. difficile while sparing the broader microbiome [11].
Microbiota Impact on Physiology
Ultimately, the platform enables personalized microbiome medicine: profiling an individual's gut ecosystem to design a bespoke LBP that restores missing functions (Cani, 2017 [12]).
References
- Murali, S. K., & Mansell, T. J. (2024). Next generation probiotics: Engineering live biotherapeutics. Biotechnology Advances, 72, 108336. https://doi.org/10.1016/j.biotechadv.2024.108336
- Optibac Probiotics. Company resources (accessed 2025).
- Behera, S. S., El Sheikha, A. F., Hammami, R., & Kumar, A. (2018). BioMed Research International, 2018, 9361614. https://doi.org/10.1155/2018/9361614
- Kleerebezem, M. et al. (2003). Complete genome sequence of Lactobacillus plantarum WCFS1. PNAS, 100(4), 1990‑1995. https://doi.org/10.1073/pnas.0337704100
- Kok, J. et al. (2005). Comparative and functional genomics of lactococci. FEMS Microbiology Reviews, 29(3), 411‑433. https://doi.org/10.1016/j.femsre.2005.04.010
- van Asseldonk, M. et al. (1990). Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis. Gene, 95(1), 155‑160. https://doi.org/10.1016/0378-1119(90)90428-T
- Steidler, L. et al. (2003). Biological containment of genetically modified L. lactis for IL‑10 delivery. Nature Biotechnology, 21(7), 785‑789. https://doi.org/10.1038/nbt840
- Wells, J. M. (2011). Immunomodulatory mechanisms of lactobacilli. Microbial Cell Factories, 10(S1), S17. https://doi.org/10.1186/1475-2859-10-S1-S17
- Steidler, L. et al. (2000). Treatment of murine colitis by L. lactis secreting IL‑10. Science, 289(5483), 1352‑1355. https://doi.org/10.1126/science.289.5483.1352
- Isabella, V. M. et al. (2018). Synthetic live bacterial therapeutic for PKU. Nature Biotechnology, 36(9), 857‑864. https://doi.org/10.1038/nbt.4222
- Kurtz, C. B. et al. (2019). Engineered E. coli Nissle improves hyperammonemia. Science Translational Medicine, 11(475), eaau7975. https://doi.org/10.1126/scitranslmed.aau7975
- Cani, P. (2017). Gut microbiota — at the intersection of everything? Nat Rev Gastroenterol Hepatol, 14, 321–322. https://doi.org/10.1038/nrgastro.2017.54