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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.

IPTG state induction speed

We have collected a huge amount of data to determine IPTG state induction time and swith change using flourometry and flux cytometry. Unfortunately we haven’t been able to analyse all of them yet. Though, we have observed that the “IPTG state” tends to impose itself over the 42ºC state in the absence of induction. We expect to show these results in the World Championship Jamboree.

Note: For an easier understanding of the bistable system we denominate "IPTG state" to the transcriptional state caused by IPTG induction; and "42ºC state" to the transcriptional state caused by 42ºC heat shock induction. In the improved flip-flop the 42ºC state causes GFP expression and the IPTG state causes RFP expression. The Basic Flip-Flop works in the opposite way: the 42ºC state causes RFP expression and the IPTG state causes GFP expression. The 42ºC/IPTG denomination will be very useful for the comparison of basic and improved flip-flops.