Revision as of 03:01, 19 September 2015

P_phoBR - I13504 - phosphate responsive promoter with GFP generator

Biology of P_phoBR

Fig.1 Phosphate sensing mechanism of P_phoBR.

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.

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_phoBR, 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_phoBR in E. coli DH10B in different phosphate concentrations

As shown in Figure 3, P_phoBR 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_phoBR is from 0-300 μM of phosphate.

Sequence and Features

References

Hsieh, Y. J., & Wanner, B. L. (2010). Global regulation by the seven-component P i signaling system. Current opinion in microbiology, 13(2), 198-203.