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Proteolysis regulation

This controlled proteolysis system could solve the problems of our bistable, since it allows a faster degradation of long-lasting proteins which are the main cause of the slowness and leakage of the system.

For this purpose, we have obtained bacteria with the Sspb gene deleted (Sspb - mutant), so that we can regulate its expression according to our needs. We have obtained this mutant and, additionally, the Clpx - mutant (mutant without the protease) directly from McGinness et al.

Our idea is to add the DAS+4 tag to the proteins of one of the states of the biestable and express Sspb under the promoter of the opposed state. Specifically, we have added the DAS+4 tag to the GFP and the LacI proteins, expressed under the cI promoter, and we have expressed Sspb under the Lac promoter (see Figure 5).

Figure 5. When the State 2 of our bistable is being expressed by IPTG (lac promoter inductor) we are not only expressing the cI repressor but we also are expressing the Sspb adaptor protein. When present, this protein binds the DAS+4 tags, that we have placed on the reporter (GFP) and State 2 repressor (LacI) both of State 1, and allows the ClpXP protease to degrade them. Therefore, when the State 2 is active we are repressing State 1 promoter and degrading State 1 reporter and State 2 repressor altogether.