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

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.

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.

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

Improvements

Team: iBowu-China 2020 Time:2020/10/26

Except the LacZ or GFP based reporting system (Part:BBa_K3225014), bio-reporters were designed for quorum sensing molecules AHLs sensing on Lux system with different features in vivo or in vitro[1][2][3]. The reporters included fluorescent, colorimetric, and bioluminescent reporter.

The reporters were tested under quorum-sensing systems in vivo with reporters of mCherry, mScarlet-I, GFP, deGFP, LacZ, and NanoLuc. Among the characterized reporters, GFP and mScarlet-I presented similar dose-response curve and LOD[2]. With the increase of time, induction fold of the two reporters improved. For LacZ and NanoLuc reporters, their LOD were evidently lower than that of the florescent reporters (3−4 orders of magnitude lower), also they showed a faster response rate (30 min for LacZ and NanoLuc and 60 min for GFP and mScarlet-I). Among all the reporters, NanoLuc provided the lowest LOD which was 3.81*10-4 nM 3OC6HSL. Notably the decrease of incubation fold after 240 min was observed for NanoLuc (table 1).

Table 1: The in vivo detection limit of reporters in different incubation time.