The Cheater Problem
In industrial bioreactors, Type-2 cheaters emerge that transcribe mRNA but fail to translate correctly, generating toxic truncated proteins that sabotage the population.
Technical details
- • Type-1 cheaters stop transcription (easier to detect)
- • Type-2 cheaters transcribe but fail to translate correctly
- • Truncated proteins consume chaperones and trigger stress
Constraints: Pristine Product, Universal QC
Current solutions require fusing tags to the target protein, altering its structure. We need a product-agnostic quality control system.
Technical details
- • No His-tags or inteins allowed
- • Must work for any target protein
- • Cannot modify the protein sequence
Dual Stress Sensors: Native Auditors
We hijack E. coli's native stress-response pathways: σ32 (cytosolic) and σE/Cpx (envelope). These act as universal auditors reporting on the cell's folding state.
Technical details
- • σ32 detects cytosolic misfolded proteins
- • σE/Cpx detects envelope/periplasmic stress
- • Both pathways are triggered by Type-2 cheaters
Production Gate: (Scyt ∨ Senv) ∧ PM
The kill logic is only armed during ProductionMode. The gate requires both stress signals AND the production inducer.
Technical details
- • Biological OR gate: either stress pathway activates
- • Biological AND gate: requires ProductionMode key
- • Prevents false positives from ambient stress
Actuator & Latch: Irreversible Commitment
The Actuator (Flp-ssrA recombinase) accumulates only during sustained stress, then flips a DNA cassette to permanently activate a toxin gene.
Technical details
- • ssrA degron filters transient noise
- • FRT sites enable irreversible DNA flipping
- • Permanent kill decision prevents cheater recovery
Translation-Completion Certificate
Good cells produce an Antidote via translational coupling—only ribosomes that finish the target protein can re-initiate and translate the Antidote gene.
Technical details
- • Antidote gene placed immediately after target stop codon
- • Dual-plug function: repressor protein + sRNA
- • Actively suppresses the kill gate in good cells
Antidote: Dual-Plug Protection
The Antidote provides two layers of protection: transcriptional repression and mRNA degradation.
Technical details
- • Repressor protein binds to kill-gate operator
- • sRNA triggers degradation of Actuator mRNA
- • Ensures only cheaters face irreversible commitment
System Partitioning for Stability
The decision core resides on the genome for stability, while swappable components are on the plasmid.
Technical details
- • Genome: Sensors, Gate, Actuator, Latch
- • Plasmid: Target protein, Certificate, ProductionMode key
- • Prevents escape via plasmid loss
Expected Outcomes & Performance Metrics
Modeling predicts ≥10× enrichment of good cells and ≤0.1% false-commit risk.
Technical details
- • Target enrichment: ≥10×
- • False commit risk: ≤0.001
- • ProductionMode threshold: tunable parameter