Programming Bacteria for Multiplexed DNA Detection

Naturally competent Bacillus subtilis (B. subtilis) can acquire DNA from the environment and incorporate new genetic information. Here we constructed a synthetic genetic circuit in B. subtilis that allows cells to detect extracellular DNA (eDNA) with specific sequences. The modular design of the circuit allows customized DNA input and gene expression output. We built living DNA sensors for detecting diverse major human pathogens including Salmonella typhimurium, Staphylococcus aureus, and Clostridium difficile, characterized sensors' sensitivity and specificity, and achieved multiplexed detection using orthogonal fluorescent reporters. Moreover, the DNA-sensing bacteria can detect target cells without DNA extraction, which is convenient for in vitro or in situ applications. We also characterized the detection efficiency of plasmid and chromosome derived from the donor cells. Plasmid can transfer to B. subtilis highly efficiently via the induction of SOS response. Specifically, plasmid can become multimerized and enhance cell lysis in donor cells, which is critical to the efficient horizontal gene transfer. The DNA transfer technology could lead to new applications in the future such as pathogen detection, disease diagnosis, and DNA processing.