Part:BBa_K546005

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

Usage and Biology

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 [http://2011.igem.org/Team:Wageningen_UR/Safety/One#Risk_Identification_of_BioBrick_System_Inside_the_Cell_Chassis 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_K546001 transformants (high copy, pSB1A2) as negative control.

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

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