Systematic design of an uncoupled arsenic-responsive bioreporter circuit in Escherichia coliView all posters
University of Lausanne, Switzerland
Synthetic Escherichia coli bioreporters for arsenic detection typically rely on the natural feedback loop that controls ars operon transcription. This loop originates because arsR, the gene that codes for the arsenic-sensitive repressor, belongs to the same operon that it controls. Feedback loops are known to show a wide range linear response to the detriment of the overall amplification of the incoming signal. While being a favorable feature in controlling arsenic detoxification for the cell, a feedback loop is not necessarily the most optimal for obtaining highest sensitivity and response in a designed cellular reporter for arsenic detection.
Here we systematically explore the effects of uncoupling the circuit input (arsenic sensing) from the output (repressor production), and develop a mechanistic model to describe relative ArsR and GFP levels in feedback and uncoupled circuitry. The topology of the arsenic sensing circuitry was changed by placing the expression of arsR under the control of a series of constitutive promoters, which differed in promoter strength, and which could be further modulated by TetR-repression, while the expression of the reporter gene was maintained under the ArsR-controlled Pars-promoter. We find that stronger constitutive ArsR production decreases arsenite-dependent EGFP output from Pars and vice-versa. This leads to a tunable series of arsenite-dependent EGFP outputs in a variety of systematically characterized circuitries. The higher expression levels and sensitivities of the response curves observed in some of the uncoupled circuits will be useful for improving field-test assays using arsenic bioreporters.