Notes

ry: Alright, so now looking at Figure 6, which shows the switching time from the high to low and low to high states of the pTAK117 plasmid.

Directing your attention to Figure 6a, in this experiment, pTAK117 cells in the low state were diluted in fresh medium and induced with 2 mM IPTG. Separate cultures were grown for 35 min to 6 h before being washed and diluted in fresh medium with no inducer. Conversely, in Figure 6b, pTAK117 cells in the high state were diluted in fresh medium with no inducer. Separate cultures were grown at 41±1C for 35 min to 6 h before being diluted in fresh medium with no inducer. (Growth was continued at standard temperature until 10.25 h after the start of the experiment and cells were assayed in the flow cytometer.)

As shown by the appearance of a bimodal distribution at 4 h (Fig. 6), the pTAK117 plasmid begins switching to the high state after 3±4 h of IPTG induction. By 5 h the switching is nearly complete, and by 6 h it is complete. On the other hand, switching from the high state to the low state is completed in 35 min or less.

You might be asking why is this the case? Well, it turns out the most important factor in determining switching time is the rate at which repressor proteins are eliminated. The switch from the low-to-high state (Figure 6a) necessitates gradual cell growth dilution (slow) of the IPTG-bound Lac repressor, whereas the switch from the high-to-low state (Figure 6b) is much quicker due to it being affected by the immediate thermal destabilization of the temperature-sensitive λ repressor. Due to these circumstances, this significant time difference is observed, and the switching to the low state is considerably faster than switching to the high state. Furthermore, the pTAK117 plasmid configuration indicates that the low state is more stable than the high one, as the rate of Lac repressor synthesis is more than an order of magnitude higher than the rate of λ repressor synthesis.