While predictable design of a genetic circuit's output is a major goal of synthetic biology, it remains a significant challenge because DNA binding sites in the cell affect the concentration of available transcription factors (TF). To mitigate this problem, we propose to use a TF that results from the (reversible) phosphorylation of protein substrate as a circuit's output. We demonstrate that by comparatively increasing the amounts of substrate and phosphatase, the TF concentration becomes robust to the presence of DNA binding sites and can be kept at a desired value. The circuit's input/output gain can, in turn, be tuned by changing the relative amounts of the substrate and phosphatase, realizing an amplifying buffer circuit with tunable gain. In our experiments in E. coli, we employ phospho-NRI as the output TF, phosphorylated by the NRII kinase, and dephosphorylated by the NRII phosphatase. Amplifying buffer circuits such as ours could be used to insulate a circuit's output from the context, bringing synthetic biology one step closer to modular design.
Keywords: Escherichia coli; NRI; NRII; genetic circuit; insulation; transcription factor.