Composite

Part:BBa_K546005

Designed by: Matthijn Hesselman   Group: iGEM11_Wageningen_UR   (2011-09-18)

Lux pL controlled LuxR + lux pR autoinducing LuxI (lva tag) + lux pR controlled GFP(lva tag).


Usage and Biology

A schematic representation of the BioBrick sub parts' interaction.

This device has three protein generators: lux pl-RBS-C0062-B0015 produces LuxR. LuxR forms a complex with the quorum sensing molecule AHL and this complex subsequently increases the transcriptional rate of the lux pR promoter while also decreasing the transcriptional rate of the lux pL promoter. Both of the other generators are under control of this lux pR promoter.

The second generator ‘luxpR-RBS-J04031-B0015’has a low basal expression of the GFP, which can - of course - be monitored.

The third generator produces luxI, which produces the quorum sensing molecule AHL. In combination with LuxR, AHL thus increases the production of AHL and GFP.

Safety Aspects

This BioBrick part produces an autoinducer that might interact with some pathogens.


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 3909
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1101
    Illegal BsaI.rc site found at 1828
    Illegal BsaI.rc site found at 2086
    Illegal BsaI.rc site found at 3189


Experimental data

This part was tested by measuring overnight E. coli cultures transformed with high copy plasmid pSB1A2 containing this insert. Fluorescence was measured in a Molecular Devices Spectramax M2 spectrophotometer, exciting the samples at 485 nm and detecting at 510 nm. 200 µL samples were analyzed in an opaque 96-wells plate. The GFP expression was monitored for 2 hours, with BBa_K546002 transformants (high copy, pSB1A2) as negative control.

The outcome is shown below. R0063-F2621 refers to the repaired version of F2621, submitted as K546003.

Fluorescence measurement BBa K546005-BBa K546002correct.png

GFP expression rapidly increases over time, indicating a functional positive feedback loop.


[edit]
Categories
//classic/signalling/receiver
//function/biosynthesis/ahl
//function/cellsignalling
Parameters
control(1) R0063 & (2) R0062 & (3) R0062
device_typefeedback
directionForward
latencySeconds
protein(1) LuxR _ (2) GFP-LVA (3) luxI-LVA
signalling_molecule3OC6HSL (AHL)
switch_point(1) 2nM