Part:BBa_K3225014

J23101-LuxR-pLux-GFP

This device is designed to detect N-(3-oxohexanoyl)-L-homoserine lactone (OHHL, quorum sensing signal molecular of E.carotovora). It consists of LuxR(quorum sensing transcriptional factor, BBa_C0062) drove by a constitutive promoter J23101 and GFP reporter drove by the binding promoter of LuxR(pLux, BBa_R0062).

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1037
  

Results

1.Results in vivo

Figure 1.The fluorescence intensity over the time with different concentration of OHHL treatment. The inducer OHHL was added to the culture after 60 min incubation.
From figure1, we found that the GFP intensity increased after OHHL incubation, even 1nM OHHL, which means LuxR could bind to OHHL efficiently. It showed that LuxR - pLux could be a highly sensitive detector of OHHL.

2.Results in vitro(cell-free system).

After the cellular sensing characterization, we tested the binding efficiency of LuxR to OHHL in cell-free systems(Fig. 3). After the overnight cell-free incubation, it showed that the circuit of LuxR-GFP was responsible to AHL with the detection limit of 1 nM.

Figure 2. The cell-free fluorescence output induced by a series of AHL concentrations. FLU was measured after overnight incubation