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Plasmids and controls

Control construction

The improved flip-flop was designed to make the control construction an easier task. By digesting the devices with the correct enzymes, we could remove or add any part anywhere within the improve flip-flop module I. The necessary control for the improved flip-flop requires two changes in the device structure: (a) removing the SspB gene from the module I to achieve a flip flop without protease activity, and (b) using an empty plasmid which provide the antibiotic resistance cassette and its possibly secondary effects instead of the plasmid with the improved flip-flop module II. The control (a) was constructed (figure 2), but analytic digestions to verify the new device are still in progress. In contrast, the control (b) was transformed with the basic flip flop in the X90 strain which was used for the experimental procedure.

Figure 2: SspB control can be obtained by digesting with Nhe I (R5) and Sal II (R6). The SspB gene would be released and the vector religated.

Vectors for improved flip-flop expression

The copy number of plasmids results to be an important factor for the function of regulatory circuits as flip-flops. Thus, we performed our fluorometry measurements with the improved flip-flop module II (asRNA) in high and low copy number plasmids. The asRNA module was cloned in a high copy number pSB1T3 vector and in a low copy pSB4A5 vector. The improved flip-flop module I was used in a medium copy number pUC commercial vector. When the improved flip-flop module II is expressed in a high copy number plasmid, the system loses its bistable behaviour, probably due to the high represion peformed by the asRNA (figure 3). Also, this module was integrated in the chromosome of the X90 SspB- and RybB- E. coli strain in single copy by using the miniTn7BB-Gm functions, but the data of these measurements are too preliminary to be shown.

Figure 3. Relative fluorescence of improved flip-flop during 42ºC induction. When the asRNA is in the pSB4A5 low copy plasmid (left) we observe an optimal behavior of the improved flip flop during the switch. In the pSB1T3 high copy plasmid (right) we can see that the relative fluorescence falls to basal levels and keeps in this way until the end of the experiment.