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Previous operations with the state-based latch (substrate showing in Fig. 4.15A) were carried out in strain DS941Z1, in which the inducible PLtet0-1 promoter is activated in the presence of aTc. Whereas, in strain DS941 which does not express the Tet repressor, the expression of Gp3 was expected to be governed only by the substrate sequence state. Therefore, when the substrate is in the RL state, Gp3 should be expressed independent of any input signal. With Gp3 expression controlled only by the state of the device, starting from the BP state a single pulse of integrase would push the device to RL state through attB×attP recombination (switch-on) reaction, switching on Gp3 and GFP expression. Then, the next pulse of integrase together with Gp3 would push the device to BP state through attR×attL recombination (switch-off) reaction, switching off Gp3 and GFP expression.

However, since the expression of Gp3 was likely to be switched on as soon as the device was pushed to RL state, initial switching maybe inhibit further switch-on reaction and promoting the switch-off reaction. It was predicted that by using a short enough pulse of inducer to activate the expression of Int for a short time, Int might be degraded before the Gp3 built up to high enough level to reverse the recombination reaction. In this way, the RL state might be reached and maintained until the next pulse of induction. This was tested by inducing strain DS941 containing substrate plasmid pZJ53off and Int expression plasmid

pZJ7 with short pulses of arabinose (half hour or one hour). After induction, the DNA states were checked by agarose gel electrophoresis (Fig. 4.15B).

Even with a short induction time (half hour), the switch-on reaction was very inefficient (about 20%) with the state-based latch in strain DS941 (Fig. 4.15B). It was hypothesized that the expression of Gp3 was very fast after the substrate DNA was recombined to RL (ON) state, which might inhibit the switch-on reaction and promote the switch-off reaction before the Int was sufficiently degraded or diluted with cell division.

To delay the expression of Gp3, a Tet repressor gene was cloned into the state-based latch substrate sequence, resulting in plasmid pZJ68off (substrate sequence showing in Fig. 4.16A). In this device, the expression of Tet repressor is on when the substrate is in BP state. The first pulse of integrase will push the device to RL state, switching on GFP expression and switching off Tet repressor expression. The residual Tet repressor is expected to repress the PLtet0-1 promoter, delaying the Gp3 expression during switching. As the Tet repressor degrades or is diluted by cell growth, Gp3 expression will be switched on, but by this point

Figure 4. 15 The switch-on reaction of state-based latch in strain DS941. (A) The architecture showing the

switching of the state-based latch in switch-on and switch-off reactions. (B) Electrophoretic analysis of the reaction. DS941/pZJ53off (BP) + pZJ7 (Int) was induced with half hour or one hour pulse of arabinose for the switch-on reaction. DNA extracted from cells after each induction was digested with SpeI (pZJ7:7175 bp, pZJ53off:1459 bp and 3917 bp, pZJ53on:2198 bp and 3178 bp) and run on the agarose gel. Percentages of DNA in RL were calculated after gel quantification.

Int will have also degraded so that the device does not reset. However, at the next pulse of integrase, there will be enough Gp3 to allow the switch-off reaction, resulting GFP expression off.

To check the set reaction on this new substrate sequence, strain DS941 containing substrate plasmid pZJ68off and Int expression plasmid pZJ7 was induced using short pulses of arabinose (Fig. 4.16B). Following the set reaction, the reset reaction was carried out using the same input pulse as used in the set reaction, to find out whether the substrate state can be restored or not in a successive operation (Fig. 4.16B).

Figure 4. 16 The switch-on and switch-off reactions of the new device in strain DS941. (A) The architecture

showing the switching of the new device in switch-on and switch-off reactions. (B) Electrophoretic analysis of the reactions. Strain DS941/pZJ68off (BP) +pZJ7 (Int) was induced in a cycle of switch-on (with half or one hour pulse of arabinose) and switch-off (with half or one hour pulse of arabinose) reactions. DNA extracted from cells after each induction was digested with SpeI (pZJ7:7175 bp, pZJ68off, BP: 2056 bp and 3917 bp, pZJ68on, RL:2795 bp and 3178 bp) and run on the agarose gel. Percentages of DNA in either RL or BP state were calculated after gel quantification.

Compared to the switch-on reaction efficiency with the state-based latch in DS941 (~20%, Fig. 4.15B), the efficiency was greatly improved with the new device (~90%, Fig. 4.16B) after either half or one hour arabinose induction. This result indicated that adding tetR into the substrate sequence increased the switch-on reaction efficiency. Meanwhile, the efficiencies greater than 60% and 50% were achieved for the switch-off reaction with the new device after the second half hour and one hour arabinose induction, respectively (Fig 4.16B).

The new device was demonstrated to change between two distinct states (high proportion of BP or RL substrate) by using a single repeating signal pulse, and it was named binary counting module. However, the best switch-off reaction (~60% efficiency with half hour induction) was not as efficient as the switch-on reaction (90% efficiency). The following sections present the experiments carried out to improve the efficiencies of the binary counting module through tuning the Gp3 expression level. It was expected that a higher Gp3 expression would be helpful for improving the switch-off reaction efficiency.

4.4.2 Optimisation of the binary counting module by changing the