• Home
• Project
• Improved Flip Flop
• Experimental Results
• Plasmids and controls

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. This control was constructed, but analytic digestions to verify the new device are still in progress.

Figure 1: 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. 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.