Composite

Part:BBa_K2240000

Designed by: iGEM2017_HKUST   Group: iGEM17_Hong_Kong_HKUST   (2017-10-18)
Revision as of 20:40, 25 October 2017 by Karencheng (Talk | contribs)


AHL sensor with positive feedback loop, GFP output and antisense RNA type 1 inhibition

The function of this part is to detect the deliberately released stimulus so as to initiate the process of 'knockout'. Considering that the released stimulus can be diluted in an environment, a positive feedback loop is introduced to amplify the signal. 3OC6HSL, which is a member of acyl-homoserine lactone (AHL) family, would be the inducer. 3OC6HSL originated from V. fischeri is a lipid molecule that can diffuse through bacterial cell membrane to facilitate the cell-to-cell communication.

This part begins with the TetR repressible promoter (BBa_R0040), which can act as a constitutive promoter of the downstream protein - LuxR (BBa_C0062) under the absence of repressor TetR. Once the 3OC6HSL is added, LuxR will form a complex with 3OC6HSL and then activates the downstream promoter, PLuxR (BBa_R0062). In the end, the production of the LuxR can be boost, thus accumulate the LuxR.

After the activation of PLuxR, LuxI protein (BBa_C0061), which is an autoinducer synthetase catalyzes 3OC6HSL from S-adenosyl-L-methionine (SAM) in cell. Due to the increase of the 3OC6HSL/LuxR complex, the entire part starting from PtetR to LuxI generates positive feedback loop, hence further induce the PLuxR. Apart from this, the 3OC6HSL molecule can also diffuse out to the extracellular environment and induce the cells nearby.

Owing to the positive feedback loop, this part would start contributing in signal emission whenever it receives 3OC6HSL molecules. As a result, it is expected to elevate the efficiency of activation under specific condition.

Considering the difficulties that previous iGEM team encountered - the leakiness of PLuxR (See experience in BBa_F2620), we tried to get rid of this by adding sequences of antisense RNA Binding regions and antisense RNA in which their interaction can counteract the activity of basal level. Ideally, this could help tackling the thorny issue which would happen when there is little expression of gene, especially without the initial activation of AHL.


Results


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1751
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1004
    Illegal BsaI.rc site found at 2455
    Illegal BsaI.rc site found at 2674


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Parameters
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