Skip to main content
. 2015 Oct 5;4:e09771. doi: 10.7554/eLife.09771

Figure 4. Novel 3-node and 5-node ring oscillators in cells.

(A) Time series traces of 3-node ring oscillators running in E. coli (mother machine). Single trap traces of 3n1 and 3n2 observed for 36 hr in vivo using a strong pPhlF sfGFP-ssrA reporter and a representative image from an ‘on’ and ‘off’ state of oscillation. Scale bar: 5 µm. (B) Time series traces of 5-node ring oscillators running in E. coli (mother machine). Single trap traces of 5n1 and 5n2 observed for 72 hr in vivo using a weak pPhlF sfGFP-ssrA reporter. (C) 3n1 displays population-wide oscillation pulses in vivo (CellASIC). Time series micrographs of 3n1 under a strong pPhlF sfGFP-ssrA reporter every 160 min; inset shows individual cells of the initial microcolony. Scale bar: 10 µm and 5 µm (inset). (D) Relationship between period and division time in vivo. Left, 3n1 in vivo under a strong pPhlF sfGFP-ssrA reporter. The in vitro data is shown for comparison. Each point in the in vivo data corresponds to the period and division time from aCellASIC experiment run under different media type and flow rates. Right, 5n2 in vivo under aweak pPhlF sfGFP-ssrA reporter. In vivo periods determined at 29ºC and 21ºC growth temperature in mother machine experiments. Boxes represent the inner quartile range with the median. (E) Influence of reporter concentration on oscillation periods by competing for ClpXP degradation. Left, with constant amounts of ClpXP the reporter concentration affects repressor degradation and thus oscillation period. Histograms of the periods observed with a weak and a strong pPhlF sfGFP-ssrA reporter for both 3n1 and 5n2 run in the mother machine. Dashed lines indicate the medians.

DOI: http://dx.doi.org/10.7554/eLife.09771.016

Figure 4.

Figure 4—figure supplement 1. Robust oscillations of 3-node and 5-node oscillators in vivo.

Figure 4—figure supplement 1.

3-node (top) and 5-node networks (bottom) oscillate with periods that depend on the network size in vivo. Shown are the distributions of observed period lengths with medians indicated by dashed lines. Both 3-node and 5-node networks exhibited robust oscillation with all growing cells oscillating for the 3-node networks and more than 95% of growing cells oscillating for the 5-node networks (defined as at least two distinct oscillation peaks per trace). Shown are four example traces for all oscillators in addition to the ones shown in Figure 3A,B. Both 3-node networks were analyzed using a strong pPhlF sfGFP-ssrA reporter and the two 5-node networks were analyzed using a weak pPhlF sfGFP-ssrA reporter.
DOI: http://dx.doi.org/10.7554/eLife.09771.017
Figure 4—figure supplement 2. Population-level oscillations of 3n2 oscillator in vivo.

Figure 4—figure supplement 2.

3n2 oscillator displays phase synchrony in vivo. 3n2 is run under a strong pPhlF sfGFP-ssrA reporter in the CellASIC microfluidic device. Scale bar: 10 µm.
DOI: http://dx.doi.org/10.7554/eLife.09771.018
Figure 4—figure supplement 3. Three-color oscillations and population-level oscillations of 3n2 oscillator in vivo.

Figure 4—figure supplement 3.

3n2 displays phase synchrony observing 3 reporters simultaneously. Reporters are a strong pPhlF Citrine-ssrA, pTetR mCherry-ssrA, and pSrpR Cerulean-ssrA. Shown is one oscillation cycle. Scale bars: 10 µm.
DOI: http://dx.doi.org/10.7554/eLife.09771.019
Figure 4—figure supplement 4. Original repressilator and OR2* mutant repressilator do not show population-level oscillations.

Figure 4—figure supplement 4.

Original repressilator and OR2* repressilator do not show phase synchrony. These are run under pTetR(r)-eGFP(ASV) in M9 minimal media; oscillations were not supported in LB. Scale bars: 10 µm.
DOI: http://dx.doi.org/10.7554/eLife.09771.020