Part:BBa_K3225003
promoter Exp
Quorum sensing is a well-known cell-cell communication system to many bacteria as well as the plant pathogenic bacteria E.carotovora. The transcriptional factors ExpRs play the same role as LuxR in the E.carotovora quorum sensing systems. ExpRs are the receptors of OHHL (a quorum sensing signal of E. carotovora). After binding to OHHLs, ExpRs can positively or negatively regulate the protein expression. ExpR153(BBa K3225002), ExpR71(BBa K3225004), and EcbR(BBa K3225005) are the OHHL receptors in three E.carotovora subspecies. And all of the three receptors can bind a promoter pExp and regulate the downstream protein expression. Here we characterized the binding and induction features of three receptors of E. carotovora. The three receptors (ExpR153, ExpR71, and EcbR) were constitutive expressed under a constitutive promoter down streamed with the pExp regulating reporters (GFP). 1) J23101- ExpR153 -pExp-GFP 2) J23101- Exp71-pExp-GFP 3) J23101- EcbR - pExp-GFP
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 209
Result
we characterized the cellular sensing features within E.coli. The engineered bacteria were incubated in the plate reader. 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 of ExpR71. However, for the bacteria with EcbR, it seemed that there was fluorescence output after 20 min incubation. And for ExpR153, the fluorescent protein was strongly expressed from the start (Fig. 1).
Figure 1. 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.
After OHHL induction, the expression of GFP was inhibited in the bacteria with the ExpR71 and EcbR. And the inhibition level was related to the OHHL concentration in the EcbR engineered host. However, there showed little inhibition or simulation in ExpR153.
The gene circuits containing three E. carotovora receptors shared the same promoter pExp. From the induction results, it can be assumed that the promoter pExp was 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. After binding to OHHL, the ExpRs negatively regulated the expression of pExp regulating genes. Among the three OHHL receptors, EcbR showed concentration-related response to OHHL.
Figure 2. The fluorescence of two engineered bacteria (EcbR and ExpR71) expressed after OHHL induction.
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