A Synthetic Genetic Circuit Enables Precise Quantification of Direct Repeat Deletion in Bacteria

Quantification of the rate of direct repeat deletion (DRD) is an important aspect in the research of DNA rearrangement. The widely used tetracycline selection method usually introduces antibiotic pressure to the tested organism, which may interfere with the DRD process. Also the length of repeat arm (LRA) is limited by the length of the TetR coding sequence. On the basis of the fluorescent microscopy and high-throughput imaging processing, here we developed a two-module genetic circuit, termed TFDEC (which stands for three-color fluorescence-based deletion event counter), to quantify the DRD rate under neutral conditions. DRD events were determined from the state of a three-state fluorescent logic gate constructed through coupling of an OR gate and an AND gate. TFDEC was applied in Pseudomonas aeruginosa, and we found that the DRD rate was RecA-dependent for long repeat arms (>500 bp) and RecA-independent for short repeat arms (