Difference between revisions of "Part:BBa K1886000"
 
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<h4>Gel electrophoretic analysis</h4>
 
<h4>Gel electrophoretic analysis</h4>
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[[File:1018luxpRLuxSDT_副本.jpg ‎|800px|thumb|left|Fig.1 Fig.1 Gel electrophoretic analyses of PCR products ]]
 
[[File:1018luxpRLuxSDT_副本.jpg ‎|800px|thumb|left|Fig.1 Fig.1 Gel electrophoretic analyses of PCR products ]]
  

Latest revision as of 00:58, 20 October 2016


pluxR->luxS

luxS is controlled by pluxR promoter. luxS is a kind of DPD synthetase, which could produce DPD (4,5-dihydroxy 2,3-pentanedione) under the cooperation of Pfs enzyme. DPD exists widely in Gram-negative bacteria and Gram-positive bacteria as the signaling factor for quorum sensing, in order to complete the information transfer process within different kinds of bacteria.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 236
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 334
  • 1000
    COMPATIBLE WITH RFC[1000]


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

Fig.1 Fig.1 Gel electrophoretic analyses of PCR products