Directed evolution of TetR for constructing sensitive and broad-spectrum tetracycline antibiotics whole-cell biosensor

Antibiotic abuse is the main reason for the drug resistance of pathogenic bacteria, posing a potential health risk. Antibiotic surveillance is critical for preventing antibiotic contamination. This study aimed to develop a sensitive and broad-spectrum whole-cell biosensor for tetracycline antibiotics (TCs) detection. Wild-type TCs-responsive biosensor was constructed by introducing a tetracycline operon into a sfGFP reporter plasmid. Using error-prone PCR, mutation libraries containing approximately 10 7 variants of the tetracycline repressor (TetR) gene were generated. The tigecycline-senstive mutants were isolated using high-throughput flow cytometric sorting. After 2 rounds of directed evolution, a mutant epS2–22 of TerR was isolated and assembled as a TCs biosensor. The epS2–22 biosensor was more sensitive and broad-spectrum than the wild-type biosensors. The detection limits of the epS2–22 biosensor for seven TCs were 4- to 62-fold lower than the wild-type biosensor (no response to tigecycline). Meanwhile, the epS2–22 biosensor had a shorter detection time and a stronger signal output than the wild type. In addition, the evolved epS2–22 biosensor showed excellent performance in detecting low traces of TCs in environmental water. These results suggest that directed evolution is a powerful tool for developing high-performance whole-cell biosensors, and the evolved epS2–22 biosensors have the potential for wider applications in real-world TCs detection.