In Experiment 1-2-1., we found that a toxin inhibits cell growth, and an antitoxin resuscitates it. However, what happens when a toxin is expressed after the antitoxin constitutive expression? Therefore, we conducted the experiment. Since cell growth was resuscitated after cells had grown, we named this experiment, "Go & Stop".

We constructed the plasmid mazF placed downstream of PBAD, the mazE downstream of Pcon, so that arabinose can induce MazF (toxin) expression. Additionally, we tried clarifying stoichiometric proportion by changing the expression of MazE with two types of RBS (B0034 and J61117) downstream of Pcon. We used E. coli transformed as follows in the experiment.

E. coli encoded MazE downstream of weak RBS (J61117) reached stationary phase with smaller turbidity and RFU of GFP than that of normal RBS (B0034). E. coli encoded mazE downstream of normal RBS (B0034) reached almost the same stationary phase as E. coli without TA system.

Fig. 3-1-2-2-3-1.

Fig. 3-1-2-2-3-2

The expression level of Prhl was so weak and the leak was high that Queen-like coli could not influence to Snow White-like coli, in other words, our final genetic circuits could not work. Taken together the results below, we re-recognated that we have to improve Prhl strength to work our final circuits.

(1) Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619
(2) Chen M. Zhang et al (2014) Distributed implementation of the genetic double-branch structure in Escherichia coli. Chinese Science Bulletin 59: 4625-4630

In Experiment 1-2-1., we found that a toxin inhibits cell growth, and an antitoxin resuscitates it. However, what happens when a toxin is expressed after the antitoxin constitutive expression? Therefore, we conducted the experiment. Since cell growth was resuscitated after cells had grown, we named this experiment, "Go & Stop".

We constructed the plasmid mazF placed downstream of PBAD, the mazE downstream of Pcon, so that Arabinose can induce MazF (toxin) expression. Additionally, we tried clarifying stoichiometric proportion by changing the expression level of MazE with two types of RBS (B0034 and J61117) downstream of Pcon. We used E. coli transformed as follows in the experiment.

E. coli encoded mazE downstream of weak RBS (J61117) reached stationary phase with smaller turbidity and RFU of GFP than that of normal RBS (B0034). E. coli encoded mazE downstream of normal RBS (B0034) reached almost the same stationary phase as E. coli without TA system.

Fig. 3-1-2-2-3-1.

Fig. 3-1-2-2-3-2

Fig. 3-1-2-2-3-3

Fig. 3-1-2-2-3-4

From this experimental result, we found that MazF inhibits cell growth and translation with MazE. Additionally, Fig 3.1.2.2.8 showed that more expression level of MazE, less likely inhibited translation of MazF stoichiometrically.
Therefore the result suggests that MazF inhibits translation, and mazE resuscitates the translation inhibition; resuscitation correlates MazE expression level. This experimental result is associated with anamnestic experimental results indicated that MazE and MazF form a hexamer (MazF2-MazE2-MazF2)
The results of Experiment 1.2.1. and Experiment 1.2.2. seem that using mazEF System can repeat the inhibition of cell growth and translation by the toxin, and resuscitation of cell growth and translation by the antitoxin.