Transcriptional Cascade

Transcriptional cascades can be seen as temporal programs of successive gene expression [4]. These gene configurations (or circuits) have the capability to integrate signals and trigger events after a delay from the initial detection event. As described before, such characteristic is needed in our project; therefore we considered two approaches based on transcriptional cascades. After some preliminary analysis, we chose to develop the approach termed negative synthetic transcriptional cascade.

Preliminary approaches

Negative transcriptional cascade

Based on the work of Hooshangi S et al 2005 [4] and the project requirements, the scheme was proposed.

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 to a signal molecule. The concentration of protein 2 will increase and achieve the threshold concentration to repress gene 3. At this point... more.

Positive transcriptional cascade

Based on the book of Uri Alone [5] and the project requirements, the scheme was proposed.

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,.... more.

Synthetic Transcriptional Cascade: The plan

Comparing advantages and disadvantages for both approaches (mainly the leaky problems), the negative synthetic transcriptional cascade was selected. After addition of IPTG, the repressor LacI will change its configuration; this will allow the transcription of TetR in plasmid 2. This molecule will act as repressor of the promoter Ptet in plasmid 2, this stops the production of the repressor cI and red fluorescent protein (RFP). After these events,.... more.

Results

Main Results

• Assembly of plasmid 1 ([http://partsregistry.org/Part:BBa_K175046 K175046]) and 2 ([http://partsregistry.org/Part:BBa_K175047 K175047]).
• Partial confirmation of sequence of both plasmids.
• Transformation in Escherichia coli
• Confirmation of both biobrick individual functionality.
• Characterization of the plasmids 1 and 2 by fluorescence experiments. ... more

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