Orthogonal Ribosome Biofirewall.

Biocontainment systems are crucial for preventing genetically modified organisms from escaping into natural ecosystems. Here, we describe the orthogonal ribosome biofirewall, which consists of an activation circuit and a degradation circuit. The activation circuit is a genetic AND gate based on activation of the encrypted pathway by the orthogonal ribosome in response to specific environmental signals. The degradation circuit is a genetic NOT gate with an output of I-SceI homing endonuclease, which conditionally degrades the orthogonal ribosome genes. We demonstrate that the activation circuit can be flexibly incorporated into genetic circuits and metabolic pathways for encryption. The plasmid-based encryption of the deoxychromoviridans pathway and the genome-based encryption of lacZ are tightly regulated and can decrease the expression to 7.3% and 7.8%, respectively. We validated the ability of the degradation circuit to decrease the expression levels of the target plasmids and the orthogonal rRNA (O-rRNA) plasmids to 0.8% in lab medium and 0.76% in nonsterile soil medium, respectively. Our orthogonal ribosome biofirewall is a versatile platform that can be useful in biosafety research and in the biotechnology industry.