Contributions

Overview

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Our 2025 team added new genetic modules, standardized wet-lab workflows, and openly shared data that expand the iGEM community’s ability to design antimicrobial peptide (AMP) probiotics. These resources cover modular nisin expression, metabolite-linked containment, and culture conditions for oral microbiome models.

Genetic & Circuit Contributions

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A. Modular Nisin Expression System

We reconstructed the full nisin operon into three compatible plasmids that co-express the maturation, activation, and export machinery. The set is designed for E. coli validation and portability to Lactobacillus with alternative promoters.

• pET-21a(+)-nisA (AmpR) — nisin precursor peptide.
• pRSFDuet-1-nisB-nisP (KanR) — modification (dehydration/cyclase recruitment) + leader cleavage.
• pACYCDuet-1-nisC-nisT (CmR) — cyclization + export.
• pMG36e_P32_comDE + PcomE→nisA (ErmR) — Constitutive ComD/ComE two-component system (P32) senses S. mutans CSP.

Intended for submission as a modular “nisin expression toolkit” with documentation for tuning promoters/RBS and secretion signals.

B. L-Asp–Responsive Kill Switch (AalR–MazEF)

We designed a metabolite-linked containment circuit that ties survival to oral L-aspartate availability: AalR (sensor) activates mazE (antitoxin) in the mouth; without L-Asp, mazF (toxin) remains unneutralized and induces RNA cleavage.

• pUPD3-AalR — constitutive L-Asp sensor.
• pET-21(+)-MazF — toxin under T7 induction window.
• pT7-MazE — antitoxin under AalR-responsive promoter.

Experimental & Data Contributions

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A. Antimicrobial Testing Workflow (Nisin)

• Standardized disk-diffusion plate setup for S. mutans lawns with clear positive controls.
• Determined commercial nisin potency was insufficient; specified recombinant purification (cation-exchange) and SDS-PAGE confirmation.

Reusable for other AMP projects with minor media/strain adjustments.

B. Culture Condition Optimization for Oral Models

• Growth curves (OD₆₀₀) for Lactobacillus, S. mutans, and E. coli at 2%, 3%, 4% glucose.
• Crystal-violet biofilm quantification across sugar levels.
• Finding: 3% glucose suppresses S. mutans growth and biofilm while maintaining Lactobacillus stability.

Datasets (growth & biofilm) prepared for download and re-analysis by future teams.

C. Noted Biological Behavior

We documented distinct growth behavior of S. mutans in liquid culture, forming visible white precipitation early in growth, unlike Lactobacillus or E. coli, which sediment only during death phase. This behavior aligns with literature describing chain-like aggregation and sedimentation linked to biofilm potential.

These observations are fully illustrated on our Results Page which includes growth curves, biofilm quantification charts, and assay photographs demonstrating the phenomenon.

Protocol & Workflow Contributions

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• Tri-plasmid assembly & antibiotic selection for nisABCTP expression.
• AMP diffusion assay (plate lawns, dose planning, positive controls).
• Crystal-violet biofilm workflow (wash → stain → wash → solubilize → A595).
• Kill-switch induction tests with L-Asp gradients and survival readouts.

Conceptual Contributions

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• Quorum-regulated AMP probiotics: on-demand expression to preserve commensals.
• Metabolite-linked containment: aligning survival with host chemistry (L-Asp).
• Oral model tuning: using sugar levels to bias against S. mutans while supporting probiotics.

Summary of Team Contributions

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Together, these contributions establish a blueprint for safe, selective AMP probiotics and provide resources that future iGEM teams can adopt and extend.