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Figure 5 | Journal of Biological Engineering

Figure 5

From: Synthetic feedback control using an RNAi-based gene-regulatory device

Figure 5

The protein-responsive OFF control device supports the design of a negative feedback control system over MS2 levels. (a) Model prediction for MS2 feedback system where miRNA level (control action) is dependent upon the level of miRNA in the cell. The output (MS2 protein) levels are shown plotted as a function of the input (maximum MS2) levels. (b) Model prediction for the MS2 system where the miRNA level (control action) is independent of the levels of miRNA. The output (MS2 protein) levels are shown plotted as a function of the input (maximum MS2) levels. (c) Schematic of the circuit used to test the MS2-responsive OFF control device exerting feedback control over MS2 levels. A doxycycline-responsive CMV-TetO2 promoter was used to control the expression of an MS2-DsRed fusion protein. An OFF control device containing an MS2-responsive ribozyme switch and a miRNA targeting DsRed were expressed in a separate expression cassette under the control of a constitutive promoter (PCMV). The expression cassettes are stably integrated into a Flp-In HEK293 T-REx cell line, which expresses the TetR repressor. Input levels (maximum MS2-DsRed levels) were varied by growing the cells under different concentrations of doxycycline. The input is highlighted in red, the output (MS2-DsRed levels) is highlighted in blue, and the control action (miRNA levels) is highlighted in yellow. (d) Output (MS2-DsRed) levels as a function of the input (maximum MS2-DsRed transcript) levels for circuits with and without a feedback controller. The output and input are reported as the percent of the maximum DsRed levels in a cell line with no miRNA present. Error bars represent the standard deviation of three independent experiments. (e) The slope as a function of the input. The local slope is calculated as: Slope i  = (Output i – Output i-1 ) /(Input i – Input i-1 ).

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