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.
Keywords: biocontainment; biofirewall; biosafety; genetically modified organisms; orthogonal ribosome; synthetic biology.