Part:BBa_K1421006

RPAI

In the photosynthetic bacterium Rhodopseudomonas palustris, there is a quorum-sensing system RPAI/RPAR, which is similar to the LUXI/LUXR system in V. Fischeri. RpaI is the acyl-HSL synthase to produce p-coumaroyl-HSL by using environmental p-coumaric acid. RPAR is a signal receptor with homology to fatty acyl-HSL receptors (such as LuxR) that responds to p-coumaroyl-HSL to regulate global gene expression including the promoter of RPAI, pRPAI.[1] In deed, there have already been numbers of quorum-sensing systems; usually, the signals of these systems are not unique and inter-influence the other. While, p-coumaroyl-HSL (pc-HSL), the signal of RPAI/RPAR system is unique to AHL. So, RPA system is independent on LUX system and a part of its homologies. This part, pRpaI, is the promoter of RPAI. It opens the expression of the gene behind and meanwhile, can be regulated by the complex RPAR/pc-HSL.

Experimantal test of RPA quorum-sensing system

Methods and Results

Constructing pSB1C3-RpaR-RPAI-GFP

1.Get GFP DNA fragments by PCR. The plasmid named pER41G-iGFP was used as template, with three primers and KOD plus working. The product was names as EGFP(P).

2.Mix pSB1C3-RpaR&I and EGFP(P) of the same volume. The next is digestion by SpeI-HF and PstI-HF(NEB). The product was named as pSB1C3-RpaR&I-GFP(P).

3.Take 5ul pSB1C3-RpaR&I-GFP for connection.

4.5ul product of connection was transformed into DH5α, the competent cell, under LB culture with Cm resistance.

5.After the extraction of plasmid, we check the results by digestion with SpeI-HF and PstI-HF(NEB). The result of DNA agarose gel electrophoresis is shown as the figure 1 on the right.

Figure 1. DNA agarose gel electrophoresis.

Analysis and Conclusion

In order to figure out what will happen to the expression of GFP if the concentration of pC-HSL is changed, we make different cultures with different concentration of pC-HSL under 37℃ with 220 rpm for 9h. The results are shown as Figure 2. When this RPA system is not induced, there is only very weak green fluorescence under UV field, which stands for the low activity of promoter of RPAI. While there will be strong green fluorescence under UV if a certain concentration of inducer pC-HSL(0.36 uM and 1.24 uM) is put into "E.coli" culture, which means a high activity of promoter of RPAI. It is clearly concluded that in constructed RPA quorum-sensing system, the promoter of RPAI is inducible and this RPA quorum-sensing system (including promoter of RPAR, RPAR, promoter of RPAI, RPAI ) works as expect.

Figure 2. Green fluorescence in E.coli with or without induction of pC-HSL using Confocal laser scanning microscopy.

Reference:

[1] 3.quorum sensing system in Rhodopseudomonas palustris:Schaefer A L, Greenberg E P, Oliver C M, et al. A new class of homoserine lactone quorum-sensing signals[J]. Nature, 2008, 454(7204): 595-599.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 80
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 199
  
• 1000
  COMPATIBLE WITH RFC[1000]