Part:BBa_K1682012

PphoA promoter

P_phoA- phosphate responsive promoter

Biology of P_phoA

Fig.1 Phosphate sensing mechanism of P_phoA.

E. coli has multiple native phosphorus sensing and regulation systems that we could use in the construct. Among them, we chose the PhoR/PhoB two-component system (TCS). It contains a sensory histidine kinase PhoR and a partner DNA-binding response regulator PhoB. PhoR is activated under low phosphate concentration, which will then phosphorylate PhoB. The phospho-PhoB is then capable of activating expression of the Pho regulon genes, two of the examples are phoA and phoBR. In high phosphate concentration, phoR is turned into an inhibitory state, which interferes with phosphorylation of PhoB. PhoB is, thus, not capable of activating expression of phoA and phoBR.

Design

The inducible promoter BBa_K1682012, created by 2015 HKUST-Rice iGEM, was originally made to control the expression of the PhoA alkaline phosphatase gene following activation by the phosphorylated PhoB transcription factor. By replacing the downstream PhoA gene with GFP, the part expresses fluorescence in the presence of low levels of extracellular phosphate.

EXPERIENCE

Utilizing the modified phosphate range and characterization protocol (See: Modified Protocol under Pho), we characterized the BBa_K3725110 phosphate sensor. However, the characterization data (Figure 1) yielded insignificant levels of fluorescence, thus leading us to conclude that the promoter is not sensitive enough to extracellular phosphate levels.

Figure 1. Characterization curve for BBa_K3725110 for phosphate concentrations between 0μM and 100μM.

Constructs for characterization

Fig.2 Phosphate sensing construct with reporter.

With the phosphate (pho) regulon from E. coli, it can be utilized for detecting phosphate level. To make a phospahte-sensing device, we obtained the promoter, P_phoA, and combined it with a GFP reporter, BBa_E0240, in BioBrick RFC10 standard so that the promoter activity in different phosphate level can be detected and characterized.

RFU measurement

Fig.3 Activity of P_phoA in E. coli DH10B in different phosphate concentrations

As shown in Figure 3, P_phoA is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 2.99 folds between 0 to 300 μM concentration of phosphate. Furthermore, a plateau is observed starting from the 300 μM phosphate concentration point, suggesting that the dynamic range of P_phoA is from 0-300 μM of phosphate.

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]