Modelling and implementation of a negative feedback mechanism in E. coli

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Nicolo' Politi, Susanna Zucca, Lorenzo Pasotti, Maria Gabriella Cusella De Angelis, Paolo Magni

University of Pavia, Italy

The interest in designing gene networks able to carry out simple tasks in a predictable way is becoming critical in the field of synthetic biology. Borrowing ideas from control systems theory, a genetic circuit which aims at maintaining a constant concentration of a signalling molecule, 3-oxohexanoyl-homoserine lactone (3OC6-HSL), was designed and implemented in E. coli-MG1655 Z1 liquid cultures. The system exploits the well-known mechanism of quorum sensing, used by the marine bacterium Vibrio fischeri to activate genetic pathways in a density-dependent manner. A bottom-up approach, based on mathematical modelling and characterization of basic parts composing the system, was used. The model includes promoters activation, enzymes production and degradation, enzymes activity and cell growth. In particular, PTetR (BBa_R0040, an anhydrotetracycline sensitive promoter) was chosen as the input of the circuit; it is responsible for the transcription of luxI, which encodes for an enzyme able to produce 3OC6-HSL. The PLux promoter (BBa_R0062) was placed upstream of aiiA, the coding sequence of a lactonase (i.e. an enzyme degrading 3OC6-HSL), so that, in the presence of a sufficiently high concentration of the signalling molecule, the transcription of aiiA occurs. In this way the system is able to self-regulate the concentration of 3OC6-HSL. First promoters activation functions were characterized, using the Red Fluorescent Protein (RFP) and varying the Ribosome Binding Site (RBS) efficiency. Then, driving the transcription rate of the genes encoding for the two enzymes through PTetR, the parameters representing the production and degradation of 3OC6-HSL were identified. In silico simulations were performed and compared to experimental data from batch culture tests: although the mathematical model well-predicts the steady state value of 3OC6-HSL concentration, it shows faster dynamics than the in vivo-implemented genetic circuit.