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Strain Construct

In order to test the improved flip flop properly, we constructed a new strain of Escherichia coli based on E. coli X90 SspB and other E. coli X90 ClpX (F’ lacIq lac’ pro’/ara [lac-pro] nalA argE [am] rifr thi-1). E. coli X90 strain has two different problems which we should solve for the accurate control of the improved flip-flop: On the one hand, RybB deletion in the chromosome was performed to achieve the expression of the RybB gene only from the improved flip-flop device. On the other hand, due to the presence of lacIq within F’plasmid, whose expression could interfere in the bistability, we caused its lost.

We have carried out the mentioned deletion to our Sspb- and Clpx- strains by using the lambda red protocol (Datsenko & Wanner, 2000). We have disrupted the chromosomal gene RybB by using PCR products in which PCR primers provide the homology to the targeted gene. In this procedure, recombination requires the phage  Red recombinase, which is synthesized under the control of an arabinose inducible promoter, temperature sensitive and low copy number plasmid. We generated PCR products by using primers with 50-nt extension that are homologous to regions adjacent to the RybB gene and the template plasmids carried an antibiotic resistance gene that was flanked by FRT (FLP recognition target) sites. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase, which is also temperature sensitive. Figure 1 shows a electrophoresis gel which demonstrates we achieved the delection in our SspB strain.

Besides, we achieved a strain without F’ plasmid by setting up a continuous culture and checking eventually if any bacteria had lost that plasmid. We observed that if the bacteria lost the F’ plasmid, they would not grow in minimal medium without proline (proline auxotrophy) and they would not turn blue in X-gal plates (lost beta-galactosidase activity).