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Stability

In order to compare the 42ºC state stability of the basic and improved flip-flop, this state was induced by harvesting bacteria cultures at 42ºC during 11 hours. This long incubation period was used to effectively achieve the cultures in the 42ºC state at the beginning of the measurements. At the “Induction Stop” point, of the graphics below (Figure X), the cultures of basic and improved flip-flop were set at 30ºC (without IPTG) and fluorometry data was taking during six and a half hours. In these graphics it is also shown how the bacteria reach the 42ºC state after change their state. 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 a MC4100 E. coli strain expressing the basic flip-flop.

Figure X. Fluorescence/O.D. of basic and improved flip-flop after 42ºC induction during 11 hours (before “Induction Stop”) and without any induction during 6,5 hours (after “Induction Stop”). 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. In the graphics we can see that the 42ºC state stability is markedly higher in the Improved Flip-Flop compared to the Basic Flip-Flop, and that the difference between the fluorescence levels of both states is also much more pronounced.

We have showed by fluorometry measurements that our improved flip-flop increases the difference between the fluorescence levels of opposite bistable states, lowering the repressed state levels to almost zero . We have also demonstrated that the stability of the improved flip-flop is much higher than the basic flip-flop under this stimulus.

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.