Part:BBa_K3989009

Quroum sensing esaS promoter: PesaS

A promoter that can interact with EsaR protein and activate downstream gene transcription.

Basic information

In contrast to P_esaRC(<a href="https://parts.igem.org/Part:BBa_K3989008"> BBa_K3989008</a>), this promoter will down-regulate the gene expression when EsaR attaches to the esa box on it. Meanwhile, this promoter has only one esa box which is at position -60.(see figure 1)

Figure 1. Mechanism of the EsaR being an activator under the control of P_esaS. The red dots are the specific AHL molecule and in our project it is 3OC6HSL[1]. When interacting with the AHL molecules, the EsaR protein will dislocate from the promoter. Thus, the P_esaS will not recruit the RNA polymerase and down-regulate the transciption.

Characterisation

Together with the EsaR protein and its variants, we characterised this promoter using the same strategy. The strategy we used is to express a GFP in a plasmid(detail of the construct: BBa_K3989025) with different EsaR protein variants. The AHL molecule's concentration are same as people used in the literature[1]. We used plate reader and flow cytometry to analyse the fluorescence generated by GFP, the results are shown in figure 3 and 4.

Figure 2. Fluorescence intensity measurement by plate reader(96-well plate). The measurements were done every one hour and this is the curve of the last test.

Figure 3. Fluorescence intensity measurement by flow cytometry. The samples are taken from the plate, in which the bacteria has been cultured for 7 hours.

From the characterisation, we reproduced the results from the literature that P_esaS can down-regulate the gene expression when the EsaR protein is dislocated from the esa box.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

1) Shong, J., & Collins, C. H. (2013). Engineering the esaR promoter for tunable quorum sensing-dependent gene expression. ACS synthetic biology, 2(10), 568-575.

2) Shong, J., Huang, Y. M., Bystroff, C., & Collins, C. H. (2013). Directed evolution of the quorum-sensing regulator EsaR for increased signal sensitivity. ACS chemical biology, 8(4), 789-795.