Team:XJTU-CHINA/Design
Design of Circuits
Precursor Circuit
Precursor circuit converts common carbon sources into geranyl diphosphate (GPP), an intermediate which will be converted into linalool and limonene by the following production circuits. Precursor Circuit contains GPPs and seven enzymes in MVA pathway which convert glucose to GPP, and all of them are IPTG inducible. To get this circuit, we reconstructed the plasmid pJBEI6409 kindly donated from 2018 team GreatBay_China which contains one more limonene synthase downstream the GPPs. Due to the lack of endogenous MVA pathway in E.coli, the co-expressed MVA pathway and GPPs will greatly improve the production of GPP.
Production Circuit
Production Circuits consist of limonene synthase (CS) and linalool synthase(LS) which enable conversions from GPP to limonene and linalool respectively. These circuits are constructed for validation of production efficiency of the CS and LS.
The limonene synthase used in our project is well studied, but since linalool synthase hasn’t been extensively characterized, so we chose three linalool synthases which are reported to exhibit high activity based on literatures, trying find the best one by their efficiency test for our further controlling circuits.
Light-Controlling Circuit
Light-Controlling Circuit is a light-stimulated gene circuit, which converts GPP in the precursor circuit to linalool or limonene depends on its state. In the presence of red light, linalool will be produced, while limonene will be produced without red light. This kind of switch can be achieved simply by physical conditions alternations.
This circuit is based on two regulation systems: Cph8 is an activator of Pomp prompter, which can be deactivated by red light (wavelength=650 nm) ; CI protein is an repressor of promoter, which will be deactivate at high temperature (42oC).
In the absence of red light, the Cph8 and OmpR complex will bind to Pomp promoter and activate the transcription and translation of limonene synthase and CI. Limonene synthase is able to convert GPP to limonene, while CI will bind to Pand block the expression of linalool synthase at 37 oC, which will repress the synthesis of linalool. In the presence of red light, Cph8 is deactivated and the expression of limonene synthase and CI will be terminated, which will further initiate the expression of linalool synthase.
Generally, the red light in our circuit will be open at night, which means linalool will be synthesized for a nice sleep while limonene will start to be produced when red light is shut down in the morning to remove sleepy mind.
Light-fluorescent Circuit
Light-fluorescent Circuits provides a series of parts for the validation of our bidirectional switch that is controlled by a photosensitive activator Cph8 and a thermosensitive repressor CI. Firstly we set a sfGFP coupling with CI under the regulation of the Pomp promoter which can be activated by Cph8, and a mRFP under the regulation of Pλ which can respond negatively to CI, and the change of fluorescent intensity of sfGFP and mRFP were detected with and without 650nm-red light, in order to quantitatively determine the responding time and peak value of these two switchable status. we also replaced sfGFP and mRFP with limonene synthase and linalool synthase respectively, in order to avoid the possible interference between GFP and RFP, and also for selective synthesis of limonene and linalool under the designed time intervals.