In silico design for Project 'Catcheater'. A modular, tag-less quality-control (QC) system for E. coli to eliminate 'cheater' cells during protein production.

Large-scale protein production is chronically undermined by "Type-2 cheater" cells. These cells mutate to produce truncated or misfolded "junk" proteins. This is disastrous for two reasons:

1. They are toxic: They trigger severe proteotoxic stress, consuming the cell's machinery.
2. They are fast: They outgrow the "good" cells, causing the entire production batch to crash.

Current solutions are flawed. "Tag-based" systems require fusing tags to the protein, which violates the need for a pristine, tag-less product. "Transcript-linked" systems are "blind" to this failure, as the cheater cell does make the mRNA.

Project "Catcheater" is a complete in silico design for a modular, tag-less quality-control (QC) "chassis" built in E. coli.

Our system hijacks the cell's native stress pathways to execute a "kill" decision, while a novel "translation-completion certificate" protects the good, productive cells.

It is built on three core components:

1. A Dual-Sensor Logic Gate: We re-wire the cell's native $\sigma^{32}$ (cytosolic) and $\sigma^{E}$ (envelope) stress sensors. This (Stress-OR-Stress) signal is fed into an AND-gate that also requires the "ProductionMode" (PM) key.
2. An Irreversible "Kill Latch": A "cheater" cell (high stress, no antidote) activates a genomic flp recombinase (the Actuator). This Flp flips a permanent DNA switch (FRT-toxin-FRT), activating a lethal mazF toxin and irreversibly committing the cell to death.
3. A "Save" Signal (The Certificate): Good cells are protected. The tetR Antidote gene is translationally coupled to the target protein's authentic stop codon. Only ribosomes that finish the protein can also make the Antidote. This tetR protein then binds to and suppresses the genomic kill-gate, granting the cell immunity.

This is the core logic of our system.

(Stress is HIGH) + (Production is ON) + (Antidote is ABSENT) → Genomic Gate (P_hybrid) = ON → flp(ssrA) Actuator is produced → mazF Latch is FLIPPED → CELL DIES

Ribosome completes protein → "Certificate" is issued (Translational Coupling) → tetR Antidote is produced → Genomic Gate (P_hybrid) = OFF (Suppressed by Antidote) → CELL LIVES

	AmirMohammad Cheraghali (Synthetic Biology and AI) - Sorbonne Université
	Sogand Azadeh (Genetics and Epigenetics) - Université Paris Cité
	AmirMahdi Karambakhsh (Systems and Synthetic Biology) - Université Paris-Saclay
	Fatemeh Sadat Mortazavi (Systems and Synthetic Biology) - Université Paris-Saclay