Preliminary Approaches

Negative synthetic transcriptional cascade

Based on the work of Hooshangi S et al 2005 (4) and the project requirements, the next scheme was proposed.

Figure 4. Genes 1 and 3 are present in the cell and they are produced all the time (black). Genes 2 and 4 (green) are in the self destructive plasmid (SDP).

Protein 1 starts the circuit activating (or repressing the repressor of) the promoter of gene 2, for proof of concept gene 1 can be changed for a signal molecule. The concentration of protein 2 will increase and achieve the threshold concentration to repress gene 3. At this point the production of protein 3 stops. As the concentration of protein 3 decreases due to its degradation, the repression of gene 4 will not longer exist and the production of protein 4 (restriction enzyme or fluorescent protein) starts. In order to make a longer or shorter delay the protein production and degradation rates have to be considered. For the former, we can “play” with the ribosome binding site (RBS) and promoter strength. The degradation can be controlled by protein stability, induction of degradation and up-down regulation of degradation enzymes.

Positive synthetic transcriptional cascade

Based on the book of Uri Alone (5) and the project requirements, the next scheme was proposed.

Figure 5. Genes 1 and 3 are present in the cell and they are produced all the time (black). Genes 2 and 4 (green) are in the self destructive plasmid (SDP).

Protein 1 starts the circuit activating the promoter of gene 2, for proof of concept gene 1 can be changed for a signal molecule. The concentration of protein 2 will increase and achieve the threshold concentration to induce gene 3. As the concentration of protein 3 increases, the induction of gene 4 (restriction enzyme or fluorescent protein) starts. In order to make a longer or shorter delay the protein production and degradation rates have to be considered. For the former, we can “play” with the ribosome binding site (RBS) and promoter strength. The degradation can be controlled by protein stability, induction of degradation and up-down regulation of degradation enzymes.

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