Journal: Nature Communications
Article Title: Robust and tunable signal processing in mammalian cells via engineered covalent modification cycles
doi: 10.1038/s41467-022-29338-w
Figure Lengend Snippet: a miRNA classifier design based on covalent modification cycles. miRNAs expected to be low in the target cell can be used to knock down the kinase, OmpR-VP64, and/or the output. miRNAs expected to be high in the target cell can be used to knock down the phosphatase, effectively increasing the output expression. Not shown for brevity, the level of OmpR-VP64 was optimized using a feedforward controller (Supplementary Figure . b Design of a miR-21 sensor for classification of HeLa cells. miR-21 knocks down phosphatase levels via 4x target sites in each of its 5' and 3' untranslated regions (UTRs). As a control, a variant was made with miR-FF4 target sites (TFF4) in place of the miR-21 target sites (T21), thus preventing knockdown by miR-21. miR-21 is differentially expressed in HeLa compared with HEK-293 cells , . c Cell type-specific signaling responses are enabled by miRNA regulation of phosphatase expression. The data is extracted from the full poly-transfection data (Supplementary Figure , comparing the second-highest phosphatase bin (P Marker ≈ 10 7 ) to the lowest (no phosphatase). d Comparison of the percent of transfected cells positive for the output for each circuit variant in HEK/HeLa cells at the optimal ratio of kinase to phosphatase markers, extracted from the full poly-transfection data. P values are from two-tailed paired T-tests between each group of samples. Receiver-operator characteristic (ROC) curves are provided in Supplementary Figures and . All data were measured by flow cytometry at 48 hours post transfection. All error bars represent the mean ± s.d. of measurements from three experimental repeats. Source data are provided as a Source Data file.
Article Snippet: The bi-normal fitting function for ROC curves is included with our MATLAB flow cytometry analysis package on GitHub (“model_ROC.mat”).
Techniques: Modification, Knockdown, Expressing, Control, Variant Assay, Transfection, Marker, Comparison, Two Tailed Test, Flow Cytometry
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