Part:BBa_K3630027
Constitutive LuxR expression and dark repression system
Figure 1 shows our Light sensor system synthesis design. Our light sensor system utilizes a light-sensitive complex Cph8 (BBa_I15010) made by the 2004 UTAustin iGEM team. This chimeric complex has three parts: The phytochrome Cph1, a transferase EnvZ histidine kinase domain, and finally a chromophore called Phycocyanobilin. This complex translocates onto the membrane of our bacteria. However, Phycocyanobilin (PCB) is not naturally produced in E. coli. Therefore, we have to express heme oxygenase 1(Ho1) (BBa_K3630013) and phycocyanobilin reductase (PcyA) (BBa_K3630014), both of which would be converting Haem (which naturally exist in E. coli) to PCB. Ho1 (BBa_K3630013) and PcyA (BBa_K3630014) are required for chromophore synthesis in photosynthetic light-harvesting complexes. In PCB pathways, Haem first undergoes cleavage towards biliverdin. Ho1 (BBa_K3630013) induces the formation of a stable Haem. HO1 (BBA_K3630013) and PcyA (BBa_K3630014) induces protein-protein interactions.
Figure 1.Cph8-OmpC mechanism)
After PCB has been synthesized, it responds to light intensity and binds to the Cph1 Phytochrome form a complex. When Cph1 absorbs light, it induces a conformational change in the complex which inhibits the autophosphorylation capability of the EnvZ histidine kinase domain. In the natural EnvZ/OmpR regulatory system, EnvZ histidine transfers phosphate to OmpR after which the OmpR will binding onto the OmpC (BBa_K3630015) promoter region to regulate the expression of downstream genes. Our OmpC promoter in the context of our system is a dark dependent promoter and will only express genes downstream of it when our BREAC is in the dark. We utilize this mechanism in our second part of the light-sensitive expression system.
Figure 2. Dark dependent repression mechanism)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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