Fig. 1: Multiparameter directed evolution of proteins in mammalian cells via robotic cell picking. | Nature Chemical Biology

Fig. 1: Multiparameter directed evolution of proteins in mammalian cells via robotic cell picking.

From: A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters

Fig. 1

a, Pipeline for multi-parameter directed evolution of proteins in mammalian cells using robotic cell picking (Supplementary Table 2). FACS, fluorescence-activating cell sorting; WGA, whole-genome amplification. b, Example data and analyses reflecting the quantitative metrics used in the cell-picker step during the second round of directed evolution, for simultaneous optimization of brightness and localization. Scale bars, 10 µm. c, Representative fluorescence images of HEK293T cells expressing the template, Archon1, and Archon2 (n = 15, 16 and 16 cells for Archon1, Archon2, and the template, respectively). Dynamic ranges for the images were normalized to facilitate visual comparison. Scale bars, 5 µm. Imaging conditions: 62 mW/mm2, λex = 628/31BP (bandpass, used throughout) from an LED, λem = 664LP (longpass, used throughout) used in c and d. d, Relative membrane localization of the indicators of c in HEK293T cells (n = 15, 16, and 16 cells for Archon1, Archon2, and the template, respectively, each from two independent transfections; ***P < 0.0001 for Archon1 and **P = 0.0003 for Archon2, Kruskal–Wallis analysis of variance followed by post hoc test via Steel’s test with the template as control group). Box plots with notches are used throughout this paper, when n > 6, as recommended by ref. 39 (narrow part of notch, median; top and bottom of the notch, 95% confidence interval for the median; top and bottom horizontal lines, 25% and 75% percentiles for the data; whiskers extend 1.5 × the interquartile range from the 25th and 75th percentiles; horizontal line, mean). e, FACS mean fluorescence intensity for sets of live HEK293T cells expressing these indicators (n = 2 transfected samples, each; individual data points in black dots). f, Representative fluorescence changes for these indicators with a 100-mV voltage step, measured in HEK293T cells. Imaging conditions: λex = 637-nm laser light, λem = 664LP, 800 mW/mm2 used for the template and 80–800 mW/mm2 used for Archons in f and g, with light intensity adjusted to prevent signal saturation. g, Population data of fluorescence changes, as in f, for these indicators (n = 5, 6, and 4 cells for the template, Archon1, and Archon2, respectively, each from two independent transfections; individual data points in black dots; error bars, s.d.; *P = 0.0155 for Archon1 and **P = 0.0374 for Archon2, Kruskal–Wallis analysis of variance followed by post hoc Steel’s test with the template as control group), taken in the steady state.