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Change Speed

42ºC state induction speed

Although the development of the improved flip-flop is focused to enhance bistability and not toogle swith speed, we compared the change speed for the “IPTG state” to the “42ºC state” transition (see the note at the end of the page) with the basic flip-flop. This assay was performed using our previously constructed X90 (SspB, RybB double deletion) E. coli strain expressing the improved flip-flop (module I and II) and the basic flip-flop. The cultures were harvesting at 37ºC overnight and the switch was force by changing the temperature to 42ºC. Green and red fluorescence levels were measured by fluorometry. The results of this assay are shown in Figure 4.

Figure 4. Fluorescence/O.D. of basic and improved flip-flop during a 42ºC continuous induction. We had to adjust the fluorescence level of the basic flip-flop, dividing the green fluorescence intensity by 7, as in this system the green fluorescence intensity is 7-fold higher than the red fluorescence intensity. This readjustment was not necessary for the improved flip-flop due to the fact that the basal intensity levels of the fluorescent proteins are similar. It can be seen that the state switch in the improved flip-flop occurs before than in the basic flip-flop. Furthermore, the graphics show a bigger green-red fluorescence difference in the improved flip-flop, compared with the basic flip-flop.