The goal of the project is to provide a proof of concept for the design and implementation of an ‘in vivo control system’ in E. coli: CTRL-E. This circuit is realized by assembling BioBrick parts with rational criteria, exploiting the information available for the basic modules (experimental data) to support a model-based approach. The circuit implementing the negative-feedback loop control is designed with the purpose to keep constant over time the concentration of the cellular signalling molecule 3OC6-HSL (involved in V. fisheri quorum sensing system), by regulating the expression of an enzyme that degrades it.
CTRL-E. is composed by two elements: a LuxI (BBa_C0061, 3OC6-HSL synthetase) expression cassette driven by the aTc-inducible pTet promoter and an AiiA (BBa_C0060, autoinducer lactonase) expression cassette driven by the 3OC6-HSL-inducible pLux promoter.
In E.coli MGZ1 strain, singled out for the case study, pTet promoter is normally repressed, due to the presence of tetR gene integrated in its genome: TetR product is able to inhibit the activity of pTet, thereby the 3OC6-HSL production. This allows the modulation of pTet activity by using tetracycline or anhydrotetracyclin (aTc) as inducers. A variation in the inducer concentration in input permits to modify the set-point of the 3OC6-HSL production in output. When a critical amount of signal molecule is reached into the cells, the complex consisting of 3OC6-HSL and its transcriptional factor LuxR (constitutively expressed by pLambda promoter) is able to activate the pLux promoter, that regulates the expression of AiiA lactonase. So the HSL molecule regulates its own production via a negative feed-back loop system.

The circuit was built assembling aiiA protein generator and luxI translational unit with BBa_K080122 composite part. Due to the length of the final circuit, the BioBrick parts were selected to reduce the internal homology, that could be cause of recombination or mutation events (since the circuit is implemented in a strain expressing recA gene). The BBa_K080122 part was purposely selected: in fact, the single terminator BBa_B1006 and the double terminator BBa_B0015 alignment does not show a significant sequence homology.
The circuit was designed without a terminator element downstream the luxI coding sequence. The lack of a terminator doesn't affect the behaviour of our circuit, since a terminator (BBa_B0054) is present in the low copy plasmid pSB4C5 used. The aiiA and luxI coding sequences are LVA tagged to decrease the protein half-life.

In order to achieve the desired system output, a fine tuning of the whole circuit is required. A deeper understanding of the transcriptional and translational strength of the regulatory elements (promoter+RBS in several combination) and of the kinetic and the activity of the involved enzymes can be exploited to identify a mathematical model able to predict the behaviour of the controlled system, in order to avoid a cost and time expensive combinatorial approach.

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