Figure 1.
Design of the expression system.
(A) PFL: the promoter CMV-TET consists of seven direct repeats of a 42-bp sequence containing the tet operator sequences (tetO), located just upstream of the minimal CMV promoter (PminCMV). The Tetracycline-controlled transactivator tTA derives from the addition of the VP16 activation domain to the transcriptional repressor TetR. The d2EYFP is the destabilised yellow-green variant of enhanced green fluorescent protein. (B) NOPFL: the CMV promoter drives the expression of the tTA, which in turns drives the transcription of the d2EYFP from the CMV-TET promoter. (Inset) RealTime PCR performed on DNA extracted from PFL and NOPLF cells shows that the DNA levels of tTA and d2EYFP are comparable among the two clonal cell populations.
Figure 2.
Degradation kinetics of d2EYFP.
CMV-TET-d2EYFP stably integrated CHO AA8 TET-OFF cells were treated at t = 0 with different concentrations of Cyclohexamide (CHX): panel A, ; panel B,
; panel C,
; panel D,
. Fluorescence intensity was followed up to 750 minutes. Sampling time is equal to 15 min. The thin line represents the mean over biological triplicates; the shaded area represents the standard error. Experimental data were used to fit the exponential decay of d2EYFP protein levels, and thus to derive its half-life (
). Fluorescence intensity in untreated cells was not subjected to any significant decay (data not shown).
Table 1.
Parameters identified after the fitting procedures: parameters values as well as standard deviation are reported for each parameter.
Figure 3.
Experimental and simulated switch off time-course across the PFL and NPFL cell population.
Experimental data (thin lines) and model simulations (thick lines) were reported for the PFL (left) and NOPFL (right) cells. Shaded areas represent standard deviations from replicate experiments.
Figure 4.
Replicates of the experimental time-courses across the PFL and NPFL cell population.
Replicates of the experimental time-courses for the PFL (left) and NOPFL (right) cells. Each line in each panel represent the average fluorescence intensity across the cell population in one switch-off experiment.
Figure 5.
Switch off time for varying Doxycycline concentrations from experimental data and model predictions.
The model predictions for the switch off times are shown for PFL (dashed thick line) and NOPFL (solid line). Experimental quantification of the
for PFL and NOPFL models have been reported for comparison with + and
respectively. Observe that the experimental
for the PFL at
and
could not be estimated since the PFL is not switching off in the experimental observation time (43 h).
Figure 6.
Phase portrait of the PFL model.
The tTA-d2EYFP mRNA concentration (y axis) has been plotted against tTA protein concentration (x axis). Varying Doxycycline concentrations ( through
) were used to investigate the dependence of the two stable equilibria (“ON” and “OFF” in the graph) on the amplitude of the input. The shape and dimensions of the two basins of attraction (the set of initial conditions ending up in one of the two stable steady states) can be studied with the same technique: in this figure the grey shaded area represents the basin of attraction of the “OFF” equilibrium for Doxycycline =
nM.