A Time-Controlled Genetic Circuit Study

Synthetic oscillators are gene circuits where the protein expression will change over time. The delay of transcription, translation and protein folding are used to form this kind of behavior. Usually, an odd number of negative feedbacks can be used to construct such oscillators. Negative feedbacks can also combine with positive feedback to constrouct, in most studys, hybrid promoters which response to both repressors and activators are used. Here, we tried to design a synthetic oscillator by a negative feedback combined with a positive feedback constructed by promoters responsing to repressor or activator.In order to construct such an oscillator, several gene circuits were designed. The candidate promoters-PR, PLux, PLac and dual-repressed promoters-were verified, and PLux, PLac together with their regulators were selected for circuits construction. SsrA-mediated tags which can reduce the half-life of proteins were added to the C-terminal of regulators to avoid protein accumulation. Two gene circuits, Os-LAA and Os-ASV, which were slightly different from each other were constructed. When glucose was used as carbon source, bacteria showed low fluorescence and fluorescence value declined during the observation. A few bacteria with Os-LAA circuit demonstrated oscillation behavior with the period about 68.3+20 minutes. When glycerol was used as carbon source, bacteria with Os-ASV circuit demonstrated high fluorescence value and oscillation behavior with fluorescence increasing. The period of oscillation was 121±21 minutes.