Overview
We advanced four research tracks toward a living, quorum-regulated probiotic therapy: culture & biofilm characterization, vector design, nisin antimicrobial testing, and killswitch safety. Each team iteratively refined its design to ensure selectivity, safety, and effectiveness.
- Determined 3% glucose as the optimal condition to suppress S. mutans growth while supporting Lactobacillus.
- Completed plasmid set for recombinant nisin biosynthesis (nisABCTP).
- Validated diffusion assay and identified low potency in commercial nisin.
- Designed a killswitch linking L-Asp sensing (AalR) to MazEF toxin-antitoxin regulation.
Culture & Biofilm Characterization
Liquid Culture — Growth Kinetics
We profiled growth of Lactobacillus, S. mutans, and E. coli in MRS with 2%, 3%, and 4% glucose. S. mutans grew slowest overall and showed the worst growth at 3% glucose (lowest stationary-phase OD). Lactobacillus and E. coli were largely insensitive to sugar concentration.
- Lactobacillus: no clear sugar preference → not sensitive to glucose level changes.
- S. mutans: slowest growth; 0-24 h log, 24-48 h stationary, >48 h death; 3% glucose = most inhibitory.
- E. coli (control): similar to Lactobacillus; no obvious preference.
Discussion: Minor deviations at 3% glucose may reflect osmotic effects or metabolite accumulation; we plan to increase biological replicates and refine sampling to improve data reliability. Notably, S. mutans formed a white precipitate early in culture — consistent with chain-like growth and sedimentation reported in literature.
Biofilm Measurement & Quantification (Crystal Violet)
After 24 h, planktonic cells were removed and wells were processed with 1×PBS → 0.1% CV → dH2O → 95% ethanol. Absorbance at 595 nm was read; media blanks were subtracted and triplicates averaged.
Result: Within the tested range, S. mutans biofilm increased with glucose concentration, whereas Lactobacillus and E. coli were largely unchanged.
Overall Outcome: Use 3% glucose for downstream co-culture and inhibition tests — it suppresses S. mutans growth and biofilm formation while maintaining Lactobacillus performance.
Vector Design
The Vector Design Team reconstructed the complete nisin biosynthetic operon nisABCTP into three compatible plasmids and verified their compatibility through antibiotic selection and restriction mapping. IPTG-inducible expression was designed for E. coli testing before transfer into Lactobacillus. Quorum-sensing and acid-responsive promoters were proposed for future self-regulation.
- pET-21a(+)-nisA (AmpR) — AMP precursor production.
- pRSFDuet-1-nisB-nisP (KanR) — post-translational modification and leader peptide cleavage.
- pACYCDuet-1-nisC-nisT (CmR) — ring formation and peptide export.
Nisin Antimicrobial Testing
Disk-diffusion assays against S. mutans were performed over multiple iterations to optimize dosage, delivery, and controls. Commercial nisin was found to have low active content, prompting the design of a recombinant purification and confirmation workflow using cation-exchange chromatography and SDS-PAGE.
| Cycle | Objective | Key Finding | Next Step |
|---|---|---|---|
| 1 | Low-dose test (50-150 IU) | No measurable inhibition | Increase concentration |
| 2 | High-dose (150-300 IU); biofilm vs non-biofilm | Biofilm inhibited; inconsistent planktonic results | Switch application method |
| 3 | Direct application | Inconsistency due to solubility/purity | Add positive control |
| 4 | Positive control (ampicillin) | Clear inhibition; validated assay | Proceed to recombinant nisin production |
Killswitch Safety
The killswitch system connects survival to oral metabolite availability using the AalR L-Asp sensor and the MazEF toxin-antitoxin pair. Each component was cloned and tuned to balance expression while maintaining containment.
- Cycle 1 (AalR): aalR cloned under p32 in pUPD3; CmR confirmed.
- Cycle 2 (MazF): mazF under T7 promoter; IPTG range optimized for control.
- Cycle 3 (MazE): AalR-responsive promoter driving mazE; safe L-Asp range defined.
- Cycle 4 (Integration): Balanced toxin/antitoxin system; ready for joint testing.
Integrated Learning
- Reconstructed plasmid system supports modular AMP production and export.
- Assay pipeline validated for consistent inhibition measurement.
- Biocontainment successfully tied to oral metabolite presence for safety.
- Culture conditions optimized for competitive probiotic advantage.
Summary of Impact
Combining AMP production, quorum-regulated control, biosafety, and culture optimization demonstrates a feasible path to a self-regulated, GRAS-based probiotic that selectively inhibits S. mutans and restores microbial balance in the mouth.
References
- Bej, A. K., Perlin, M. H., & Atlas, R. M. (1988). Model suicide vector for containment of genetically engineered microorganisms. Applied and Environmental Microbiology, 54(10), 2472–2477.
- Mai, H. T. X., Van Hau, N., Nghia, N. H., & Thao, D. T. P. (2018). Expression and Purification of Nisin in Escherichia coli. International Journal of Life Sciences Scientific Research, eISSN 2455-1716, 1716.
- Mierau, I., & Kleerebezem, M. (2005). 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Applied Microbiology and Biotechnology, 68(6), 705–717.
- Shin, J. M., Gwak, J. W., Kamarajan, P., Fenno, J. C., Rickard, A. H., & Kapila, Y. L. (2016). Biomedical applications of nisin. Journal of Applied Microbiology, 120(6), 1449–1465.
- Wasfi, R., et al. (2018). Probiotic Lactobacillus sp. inhibit growth, biofilm formation and gene expression of caries-inducing Streptococcus mutans. Journal of Cellular and Molecular Medicine, 22(3), 1972–1983.
