A fast, robust and tunable synthetic gene oscillator - The BioCircuits ...

doi: 10.1038/nature07389 SUPPLEMENTARY INFORMATION
prewarmed. Between 2.5–4 h after initial dilution, aliquots of the growing culture were added
to inducer tubes at regular intervals, resulting in a set of cultures with varying induction times.
At the end of the experiment, all samples were quickly measured by flow cytometry (∼3 samples/min),
starting with the lowest induction duration. The induction duration was calculated
as the time between addition of culture to inducer and flow cytometry measurement, and the
oscillatory period was calculated as the time between the first peak and the second peak of the
mean fluorescence of the culture.
This protocol allowed parallel examination of multiple inducer conditions more easily than
the continuous timecourse protocol. Amplitudes and oscillation periods increased along the
arabinose transect at 2 mM IPTG as arabinose increased (Supplementary Fig. 9). At low levels
of arabinose, mean fluorescence was low enough that following oscillations after the second
peak became difficult. However, microscopy experiments at these arabinose levels confirmed
that the cells were oscillating. The values seen along the IPTG transect at 0.7% arabinose were
in good agreement with the period measurements obtained from single-cell trajectories (Fig. 2a),
although measurements derived from single-cell trajectories were consistently higher. This could
be due to an unexplained difference in the growth environment (although doubling times were
consistent across the two methods), or it could be related to the gradual increase in period seen
in the microscopy experiments (Supplementary Fig. 4). Extension of the IPTG transect to IPTG
levels higher than those examined by microscopy resulted in oscillations with low amplitudes
and short periods, confirming the decrease in period seen in the microscopy experiments.
Avg FL (AU)
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Time after induction (min)
Supplementary Figure 9: Oscillations in mean fluorescence over time on a transect along 2 mM IPTG.
Fluorescence was measured by flow cytometry of samples induced with various levels of arabinose (blue,
0.2% arabinose; green, 0.3%; orange, 0.7%; red, 2%) and 2 mM IPTG and grown for a range of durations,
and smoothed by a Savitsky-Golay filter. (Flow cytometry data for the 2% arabinose sample are shown in
Supplementary Figs. 14–15.)
Hybrid promoter induction curves were determined using MG1655Z1 cells (Lutz and Bujard
1997) transformed with a plasmid that contained yemGFP-ssrA under the control of the p lac/ara-1
hybrid promoter (essentially pJS167 without the activator module). MG1655Z1 constitutively
produces LacI, which is necessary to support IPTG-mediated release of repression given the large
number of lacO sites on multiple-copy plasmids. AraC is produced from the wildtype genomic
locus. These cells have neither feedback loop from the dual-feedback oscillator. An overnight
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