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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, this is not our ultimate goal. We also designed sgRNA targeting to luciferase,which offers us a way of quantitive test of our system, and TesA, which is one of the vital enzyme 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 green light-sensing system sharing a common chromophore, we construct 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 integration of promoter, Cph8 sequence and the terminator is then cloned into pCDFDuet. At the same time, sgRNA, which specifically taget on RFP gene, is also inserted to the same plasmid however invertedly.
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.
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