Fig. 1
From: Bacterial variability in the mammalian gut captured by a single-cell synthetic oscillator
RINGS analysis measures single-cell repressilator 2.0 phase. a The repressilator is a ring-oscillator comprising three repressors. b Colonies expressing the repressilator 2.0 display fluorescent rings controlled by the circuit’s oscillations. c Repressilator phase progresses based on bacterial divisions, with an ~15 generation period. The phase of the colony-initiating bacterium at the time of plating (θ0) controls the position of the fluorescent rings forming within that colony. d The RINGS pipeline consists of plating of a bacterial population on agar plates, followed by imaging, computational identification and export of individual, centered colonies and fitting of a generative model. The fit of ring position is then used to estimate θ0 at the time of plating. e LPT239 bacteria, carrying the repressilator, are synchronized by exposure to aTc or IPTG as demonstrated by average projections of aligned colony images compared to unsynchronized controls. Scale = 0.1 cm. e RINGS analysis demonstrates the ability of the repressilator to report on bacterial phase. Graph shows polar histogram representing normalized counts of colonies (radius) within a given θ0 range (angle). For e, f IPTG n = 93; aTc n = 62; unsync n = 49. g LPT320 bacteria were synchronized with aTc and grown in log-phase growth at 37 °C, plating samples for colony counts and RINGS analysis every 2 h ± 15 min. h RINGS analysis measured phase (θ0) across the population. Graphs show histograms of normalized θ0 count for two biological replicates. Arrows indicate shift in population since last timepoint i. Repressilator phase correlates with the estimated generations as determined by CFU counts from agar plating. Graph shows mean with 95% CI (error bars smaller than datapoints) of elapsed phase after removal of aTc. j Cumulative distribution functions of predicted generations elapsed across the population. Labels list average generations for each timepoint as calculated by CFU counts in panel i. For h–j, the number of colonies analyzed are listed in j. Source data are provided as a Source Data file
