Fig. 4: Functional optogenetics in K. rhaeticus.
a, Proposed procedure to make patterned BC through optogenetics. b, The Opto-T7RNAP system uses a split T7-RNA polymerase, made blue light activatable via fusion with photo-sensitive magnet proteins. c, Genetic arrangements of K. rhaeticus optogenetic strains. Expression of the split T7RNAP genes is induced with arabinose. d, Red fluorescence scan of the top surface of a blue light exposed wet K. rhaeticus pOpto-T7RNAP*(563-F2)-mCherry pellicle (diameter = 150 mm). Graphic on top right shows the image projected onto the pellicle during growth. Pellicle shown is representative of two patterned pellicle repeats. e, The right of the projected image contained a gradated strip, from minimum to maximum light let through. Data show the intensity of red fluorescence seen in the pellicle against this gradated strip. The s.d. of pixel intensity for each horizontal slice is shown in pink. Black dotted line represents the intensity of unexposed pellicle regions. f, Smallest projected mark on the exposed pellicle. g, Characterization of optogenetics constructs with mCherry target gene under differing arabinose percentage (wt/vol) concentration. Bars (blue, exposed and gray, unexposed) show mean increase in red fluorescence after 6 h normalized by OD600. Error bars show s.d. of three biological replicates. Fold difference between exposed and unexposed cells is shown above, except in instances of poor growth. h, Comparison between projection video and the resulting wet K. rhaeticus Opto-T7RNAP(563-F1)-tyr1 pellicle after eumelanin development (dimensions = 300 × 170 mm). Rectangles (black and blue) at top of projection video are timed to appear to aid in calculating minimum light exposure time. Densitometry scan and photograph of top surface of pellicle are shown. i, Zoomed-in sections of a densitometry scan of the K. rhaeticus Opto-T7RNAP(563-F1)-tyr1 pellicle. Black triangle points to the sixth rectangle, indicating a 40-h required exposure time. j, Optogenetic construct characterization with tyr1 under differing arabinose induction. The bars (blue, exposed and gray, unexposed) show the mean and s.d. of three biological replicates of initial (0–100 min) reaction rate of eumelanin production measured at OD405, normalized to the number of initial cells at OD600 at time point 0.
