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. fischeri 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_K081022 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_K081022 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.

Four basic components of this circuit were identified as crucial to assess the desired circuit behaviour: the promoters pLux and pTet and the enzymes luxI and aiiA.
For each part, a simple measurement system was designed, built and tested, to gather more information about its functioning.
All modules were tested in E.coli MGZ1 strain.
More in detail, the promoters were tested with four different RBSs (RBSX stands for one of these BioBrick parts: BBa_B0030, BBa_B0031, BBa_B0032, BBa_B0034) upstream of an mRFP coding device. The enzymes were assembled under the control of pTet promoter and HSL was measured (using T9002 biosensor – see Modelling section) to determine the degradation or synthesis kinetics.

Image of pLux-RFP (with LuxR!!)

Image of pTet-AiiA

Note: Whilst in the final circuit aiiA expression is regulated by pLux promoter, in the measurement system it is driven by pTet promoter in order to avoid interference between inducer and gene product.

Image of pTet-LuxI