How?

From the literature review, two different genetic circuit configurations were contemplated: A synthetic transcriptional cascade, which has been showed to perform time-delay behavior in previous studies (4) and an approach based on post-transcriptional regulation which we termed biosynthetic AND gate.

As the conjugation system will have two plasmids (conjugation and helper plasmids), the approach followed in this sub-project was to split the construction of the time-delay genetic circuit in two independent plasmids which in theory should be present in a single cell in order to initialize the time-delay. This can be achieved given two different selection markers and apply the two different selection pressures.

Due to the expected long delay time needed, an optimistic approach will be the combination of both approaches, synthetic transcriptional cascade and biosynthetic AND gate in order to gain a desire phenotype.

Figure 3. Time-delay genetic circuit approach. Two approaches have been considered: 1) (Blue circle) Synthetic transcriptional cascade, the delay is due to a sequential expression of genes, and 2) (Green circle) Biosynthetic AND gate, the delay is due to the need of the presence of two different events and post-transcriptional control. In order to achieve a long delay both approaches could be ultimately combined.

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