Latest revision as of 05:18, 7 October 2022

PphoA promoter

P_phoA- phosphate responsive promoter

Biology of P_phoA

Figure 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The inducible promoter BBa_K1682012, created by 2015 HKUST-Rice iGEM, was originally made to control the expression of the PhoA alkaline phosphatase gene following activation by the phosphorylated PhoB transcription factor.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.

Constructs for characterization

Figure 3. 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

Figure 4. 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

Prairie_iGEM2022_UManitoba

Summary

The 2022 UManitoba iGEM team uses this part, along with BBa_B0032, BBa_K3033009, and BBa_B0015 to create a sensor to detect presence of phosphate based on fluorescence of EGFP (BBa_K3033009). Different concentrations of phosphate are tested to identify a suitable concentration range. The sensor is cloned into three different backbones (pUCIDT and pSB1C3) to optimize EGFP expression.

Characterization in pUCIDT with different concentrations of phosphate

BL21(DE3) cells transformed with a phosphate sensor on pUCIDT backbone are used for testing. Expression in Erlenmeyer flask with 0 uM and 500 uM sodium phosphate. For data below, normalized fluorescence intensity according to an optical density at 600 nm (OD600) are reported.

Figure 1. Expression of EGFP detected in phosphate deficiency condition.

Figure 2. The fitted curve of EGFP expression in response to phosphate for PhoA promoter.

Conclusion

From our result, we can conclude that this phosphate sensor part can be used to detect phosphate at below 100 uM phosphate when this part is used with a high copy number plasmid such as pUCIDT.