Part:BBa_K1893001

Las receiver with GFP reporter (LasR+pLas+GFP)

The gene encoding the transcriptional activator LasR is downstream a constitutive Anderson promoter, j23101. LasR is activated by 3O-C12-HSL. Included in the construct is the LasR-activated promoter pLas upstream of a reporter sequence including GFP. This part was designed so that we could characterize the LasR quorum sensing system and determine activation ranges and crosstalk data.

Characterisation data

Figure 1. Characterisation of the Las response device (BBa_K1893001). (A) Transfer function curve of normalised fluorescence against cognate inducer C12-AHL (3O-C12 AHL) concentrations. (B) Heat map of normalised fluorescence of RhlR-GFP system over a range of AHL concentrations: (i) Binding of RhlR-GFP to its cognate AHL (3O-C12 AHL). (ii) Binding of RhlR-GFP to 3 non-cognate AHLs (C4 AHL, 3O-C6 AHL, 3OH-C14 AHL). (C) Transfer function curves of normalised fluorescence against non-cognate inducer AHL (3O-C12 AHL) concentrations to investigate inducer AHL crosstalk: (i) C4-AHL of the Rhl system (ii) C6-AHL (3O-C6 AHL) of the Lux system (iii) C14-AHL (3O-C14 AHL) of the Cin system. Experiments were performed in E. coli Top10 cell strain cultured at 37°C. Normalised fluorescence was calculated by dividing fluorescent signal by cell density (OD600). Fluorescence measurements were recorded at 180 minutes. Reported values represent the mean normalised fluorescence value from 3 technical repeats and error bars represent standard deviation of these.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 383
  Illegal AgeI site found at 580
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1719