Team:iBowu-China/Demonstrate
- We characterized four quorum sensing system for the pathogenic detection, and selected a sensitive E. carotovora OHHL sensing system.
- We have developed a cell-free quorum sensing signal OHHL detection platform.
- We established a high-sensitivety test paper for the detection of soft rot.
Our experimental study is mainly divided into five parts, and the experimental characterizations of our detection systems are shown as follows.
We selected four bacterial quorum sensing systems for the E. carotovora pathogenic detecting and controlling. Three quorum sensing signal (OHHL) receptors of E. carotovora and the common known receptor LuxR were chosen for the soft rot sensing elements. ExpR153, ExpR71, and EcbR are the OHHL receptors of three E. carotovora subsp. respectively. And LuxR is the AHL receptor of Vibrio fischeri. The sensing gene circuits were constructed as the following design (Fig. 1):
- pcon - ExpR153 -pExp-GFP
- pcon - ExpR71R-pExp-GFP
- pcon - EcbR - pExp-GFP
- pcon - LuxR- pLux-GFP
- pcon - ExpR153R -pExp- LacZ
- pcon - Exp71R-pExp- LacZ
- pcon - EcbR - pExp- LacZ
- pcon - LuxR- pLux-LacZ
The four receptors (ExpR153, Exp71, EcbR, and LuxR) were constitutive expressed under a constitutive promoter down streamed with the pExp/pLux regulating reporters (GFP and LacZ).
The construction of circuits was completed by Gibson Assembly, and confirmed by sequencing.
Figure 1. Schematic diagram of gene circuits. The four receptors (ExpR153, Exp71, EcbR, and LuxR) were constitutive expressed under a constitutive promoter down streamed with the pExp/pLux regulating reporters (GFP and LacZ)..
After the construction of the circuits, we first characterized the cellular sensing features within E.coli. The circuits were transformed into E.coli. It can be visible in green because of the high expression of GFP after incubation for a period of time. The engineered bacteria were incubated in the plate reader, and OD600 and fluorescent intensity were measured for cell density every 5 min for two hours. During this period, the inducer OHHL was added after 1 h.
Before OHHL induction, it showed that there was no expression of the engineered bacteria with gene circuits of pcon-ExpR71-pExp-GFP and pcon-LuxR-pLux-GFP. However, for the bacteria with pcon-EcbR-pExp-GFP, it seemed that there was fluorescence output after 20 min incubation. And for pcon-ExpR153-pExp-GFP, the fluorescent protein was strongly expressed from the start.
After OHHL induction, it was shown that the fluorescent protein was expressed within the circuit of LuxR. And 1 nM OHHL could cause the regulated expression. However, the fluorescence protein expression was inhibited in the bacteria with ExpR71 and EcbR.
The gene circuits containing three E. carotovora receptors shared the same promoter pExp. From the induction results, we assumed that the promoter pExp was only activated by the free receptors. The fluorescence results showed that the binding affinities of ExpR153 with promoter were stronger than those of EcbR, and the ExpR71 was the weakest. On the contrary, the pLux were activated by the LuxR binding with OHHL. In general, the inducer OHHL was positively regulating the down streamed expression of LuxR based operon and negatively regulating of the ExpR based operon. Therefore, we chose the positive regulation quorum sensing system (LuxR-pLux) for the following detection experiments.
Figure 2. The fluorescence of engineering bacteria with four gene circuits in the 100 min incubation. The inducer OHHL was added to the culture after 60 min incubation.
Figure 3. The fluorescence of three engineered bacteria expressed after OHHL induction.
After the cellular sensing characterization, we applied our gene circuits to the cell-free systems. The gene circuits were characterized in cell-free system with a series of inducer (OHHL) concentrations. After the overnight cell-free inducing incubation, it showed that the circuit of LuxR-sfGFP was response to OHHL with the detection limit of 1 nM. In this detection system, no more than 10 nM of OHHL could induce the strong fluorescence output (Fig. 4).
Figure 4. The cell-free fluorescence output induced by a series of OHHL concentrations.
Based on the cell-free induction results, we placed the quorum sensing system to papers by freeze-drying technology. The reporters were replaced by the visible signal protein LacZ. LacZ is β-galactosidase which can cleave the yellow substrate to purple. The four gene circuits were tested to OHHL inducing experiments.
Similar to the previous results, only the LuxR-LacZ gene circuit was successfully detecting the OHHL. Among the series of OHHL concentrations induction, it showed that the detection limit was lower than 1 nM. Moreover, there is a significant visible output with 10 nm OHHL treatment (Fig. 5).
Figure 5. The detection limit and detection time of quorum sensing based testing strips.
When we got the high-sensitivity test paper for Erwinia carotovorum, we hope to develop bio-pesticides based cell-free systhesis that can prevent soft rot. We plan to express the hydrolase AiiA and the antibacterial peptides will cell-free system which could degrade AHL and generally kill bacteria separately.