Model-guided design for ultrasensitive and multiplexable cell-free diagnostics

Cell-free systems offer practical and technical advantages over whole-cell sensors for point-of-use detection of water contaminants like arsenic, mercury, fluoride, and nitrate. However, the diversity of sensors that can function in E. coli extracts is constrained by the scarcity of characterized strong promoters that can be regulated by allosteric transcription factors. Because engineering promoter strength without affecting inducibility remains an unsolved challenge in synthetic biology, the output signals from cell-free sensors are often undesirably low, particularly when detecting trace contaminants.

To address this problem, we have designed a new scheme in which the output from a cell-free sensor is amplified using an intermediate bacteriophage RNA polymerase synthesized in situ. Our experimental results are guided by an ordinary differential equation model for cell-free gene expression that predicts cascaded amplification improves the sensor’s detection limit and generates tunable ultrasensitivity. Positive feedback introduced through autocatalytic transcription and translation further accelerates the time required for a visible signal. By deploying orthogonal polymerases in parallel, four key water contaminants should be detectable simultaneously, with high ON signal, in a single pot. We anticipate that this work will have transformative impact toward the engineering of highly sensitive and field-deployable cell-free biosensors for monitoring global water quality.