Part:BBa_K1886002
AHL,DPD double-cycled oscillation
Two molecules of luxR binds with AHL (catalyzed by luxI), and this compound will promote the activity of pluxR. pluxR controls the expression of gene aiiA and luxS, Gene aiiA encodes the enzyme which degrades AHLs. And gene luxS produces DPD. Therefore, we could realize an AHL-DPD double-cycled oscillation, and the phase difference is half a cycle.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1821
Illegal BamHI site found at 4352 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 3256
Illegal AgeI site found at 4450 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1101
Illegal BsaI.rc site found at 1970
Illegal BsaI.rc site found at 3073
Illegal BsaI.rc site found at 4114
Characterization
BACKGROUND
Overview
The difference between the part for single-cycled oscillation and the part for double-cycled oscillation is that pluxR and LuxS are added, which enable the connection difference of the expression of AHL and DPD. The expression quantity of DPD and aiiA will increase due to the accumulation of AHL, while the increased connection of aiiA will hold up the expression of AHL in return. In that way, the peak value of these two substance is staggered.
Principle
The ZJU-modified synchronized oscillator design is based on elements of the quorum sensing machineries in Vibrio fischeri and Bacillus Thurigensis. We placed the luxI (from V. fischeri), aiiA (from B. Thurigensis) and DPD which is another auto inducer widely existing in Gram-negative bacteria and Gram-positive bacteria under the control of three identical copies of the luxI promoter. The LuxI synthase enzymatically produces an acyl-homoserine lactone (AHL), which is a small molecule that can diffuse across the cell membrane and mediates intercellular coupling. It binds intracellularly to the constitutively produced LuxR, and the LuxR–AHL complex is a transcriptional activator for the luxI promoter. The expression of the auto inducer DPD increases. AiiA negatively regulates the promoter by catalysing the degradation of AHL. This network architecture, whereby an activator activates its own protease or repressor, is similar to the motif used in other synthetic oscillator designs and forms the core regulatory module for many circadian clock networks. Furthermore, theoretical work has shown how the introduction of an autoinducer in similar designs can potentially lead to synchronized oscillations over a population of cells. These two auto inducers can trigger different promoters when their expression quantity reaches a certain value, which means different circuits will be triggered, then different downstream proteins will be expressed. Therefore the execution of different signal during different time is realized.
RESULTS
Gel electrophoretic analysis
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