Many kinds of plant and bacteria use a class of photoreceptor proteins, phytochromes to control phototaxis, photosynthesis and the production of protective pigments. A phytochrome found in cyanbacterium has been engineered and wildly used as light sensing system in E.coli to control gene expression since 2005. Considering light could be applied with high resolution across space and over time, we choose this powerful tool to manipulate gene expression quantitively by changing wavelength and intensity.

Red Light-Sensing System

The red light-sensing system we chose relies on one important synthetic sensor kinase, Cph8. By fusing phytochrome Cph1 from cyanbacterium and histidine kinase EnvZ from E. coli, this chimeric light receptor is engineered and expressed in a phosphorylated ground state with the help of chromophore. Besides, there are two pigment molecules, hoI and pcyA, found to be of vital importance during the process of the generation of the chromophore phycocyanobilin (PCB). Interacting with PCB, Cph8 passes a phosphoryl group to OmpR, which then initiates downstream gene transcription.

Once inactivated by red light (650nm), Cph8 is switched to an unphospholated state so that OmpR stays unphospholated as well. Hence downstream gene cannot be transcribed.

=Downstream Regulation= With a '''OmpC''' promoter, we inserted the sgRNA downstream in an opposite direction, avoiding the interference between transcription of sgRNA and cph8. This sgRNA is designed to '''target the upstream sequence on RFP'''. In the '''absence of red light''', sgRNA will be expressed and thus '''RFP will be repressed'''. Once '''treated with red light''' (650nm) continuously, the expression quantity of '''RFP will rise''' as light intensity increases. However, we cannot directly use RFP as a quantitive indicator, because the wavelength of exciting light of the red sensor is quite close to the wavelength of RFP. Thus, avoiding the interference brought by exciting and indicator light, we also designed the sgRNA targeting the upstream sequence of '''luciferase''', so that we could get '''quantitive data''' while treating with light of different intensity. However, this is not our ultimate goal. We also designed sgRNA targeting to '''TesA''', which is one of the vital enzymes in fatty acid metabolic pathway. Because of the characteristic of sgRNA, we could design various controlling systems to realize our regulation on genome by simply changing the base-pairing region of sgRNA. =Sensor Design= Considering '''red and [http://igem.bio-x.cn/wiki/index.php?title=Team:SJTU-BioX-Shanghai/Project/Light_sensor/Green green light-sensing system] sharing a common chromophore''', we constructed two different plasmids expressing '''pigments and light sensor separately'''. We cloned the Cph8 sequence into modified pSB1C3 in order to integrate it with a more stable and uniform expressed constitutive promoter. The integral of promoter, Cph8 sequence and the terminator was then cloned into pCDFDuet. At the same time, sgRNA, which specifically taget on RFP or luciferase gene, is also inserted to the same plasmid however '''invertedly'''. However, during the process of construction, we found that it difficult to get correct clone when screening. After discusion, we assumed that the bacteria was under great pressure when so many constant expressing genes was transferred into. So we also constructed the plasmid with cph8 promoted by '''T7 promoter''', in order to sellect an appropriate constitutive promoter with suitable strength. [[File:A123456.png|center|600px]] [[File:A1234567.png|center|600px]]

References

LEVSKAYA, A., CHEVALIER, A. A., TABOR, J. J., SIMPSON, Z. B., LAVERY, L. A., LEVY, M., DAVIDSON, E. A., SCOURAS, A., ELLINGTON, A. D., MARCOTTE, E. M. & VOIGT, C. A. 2005. Synthetic biology: engineering Escherichia coli to see light. Nature, 438, 441-2.

TABOR, J. J., LEVSKAYA, A. & VOIGT, C. A. 2011. Multichromatic control of gene expression in escherichia coli. Journal of Molecular Biology, 405, 315-324.