Biological Modules

Input

For the input, it was needed to use a signal that can easily represents an “ON and OFF” switch. The use of lights to represent the inputs was our first idea: a blue and a red light.
In the final E.calculus design, each sensor enables transcription of a specific recombinase (TP901 for the red sensor and Bxb1 for the blue sensor).

Blue light system
The blue light sensor uses the couple YF1-FixJ/FixK2 promoter. This couple was already used by iGEM11_Uppsala-Sweden team and described in literature (Andreas Möglich et al. 2009. Design and Signaling Mechanism of Light-Regulated Histidine Kinases.). YF1 is the fusion of the LOV protein and an histidine kinase. With no light, YF1 can phosphorylate FixJ and the phosphorylated FixJ activates the PfixK2 promoter. In presence of a 480 nm wavelength light, YF1 can no longer phosphorylate FixJ and there is no activation of the PfixK2 promoter.
''Blue light system characterization''
To characterize the blue light sensor, a modified RFP was used as the output instead of the recombinase Bxb1. In darkness, RFP is supposed to be express whereas in presence of blue light, no RFP is transcripted.

Red light system
The red light sensor uses CpH8, a chimeric receptor interacting with the promoter OmpC. CpH8 is the fusion of PCB photoreceptor and EnvZ histidine kinase (essential to be used in E.coli deficient in wild-type EnvZ). PCB requires biosynthesis of two genes : ho1 and PcyA. With no light, Cph8 is phosphylated and can activate the ompC promoter. In presence of a 660 nm wavelength light, cph8 is no longer phosphoryalated and can not activate the ompC promoter.
''Red light system characterization''
The system is similar to the blue light sensor, a modified RFP was used as the output instead of the recombinase TP901. In darkness, RFP is supposed to be express whereas in presence of red light, no RFP is transcripted. We succeed to obtain the construction but needed time to characterize it. More details here : Results