Introduction

Phosphorus plays an essential role in plant growth. It associates with various growth factors for root development, seed production, etc. Deficiency in phosphorus leads to stunted plant growth, yet the symptoms are not obvious. Therefore, it is important to monitor the levels of phosphate in the soil for healthy plant growth.

Phosphate sensor Design

Escherichia coli (E. coli) detects inorganic phosphate (P(i)) from the environment by the PhoR/PhoB two-component system (Hsieh & Wanner, 2010). As illustrated in Figure 1, P_phoA and P_phoBR is cross-regulated by PhoB and PhoR. The sensory histidine kinase PhoR behaves either as an activator or inactivator for PhoB depending on different states (inhibition state, activation state, deactivation state). When phosphate is limited, PhoR act as a phospho-donor for the autophosphorylation of PhoB. The phosphorylated PhoB will directly activate P_phoA and P_phoBR. In contrast, when there is phosphate, PhoR interferes with phosphorylation of PhoB which in turn inactivates both P_phoA and P_phoBR.

With the phosphate (pho) regulon from E. coli, it can be utilized for detecting phosphate level. P_phoA and P_phoBR from E. coli strain DH10B was cloned and ligated with the GFP generator (pSB1C3-BBa_I13504) respectively. Under high phosphate concentrations, repression on the green fluorescence intensity is expected; while under low phosphate concentrations, expression on green fluorescence is expected.

Experiments performed

Characterization on the constructs (BBa_K1682013) was done using M9 minimal medium (without phosphate). Quantitative characterization on the promoters were done by measuring the fluorescence signal intensity using an EnVision® multilabel reader.

The results were obtained by combining the 3 characterization results together.

Please visit Phosphate sensor Experiment Protocol for more details.

Results

After measuring the GFP signal intensity using an EnVision® multilabel reader, the data were processed in Relative Fluorescence Unit (RFU) (in OD600) against phosphate concentration.

As shown in Figure 2a, P_phoA is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 2.99 folds between 0 to 200μM concentration of phosphate. Furthermore, a plateau is observed starting from the 200 μM phosphate concentration point, suggesting that the dynamic range of P_phoA is from 0-200 μM of phosphate.

As shown in Figure 2b, P_phoBR is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 1.88 folds between 0 to 250 μM concentration of phosphate. Furthermore, a plateau is observed starting from the 250μM of phosphate concentration point, suggesting that the dynamic range of P_phoBR is from 0-250μM of phosphate.

Discussion

In order to select a better phosphate sensor, the fold change of P_phoA and P_phoBR were being compared.

Figure 3. Fold change of P_phoA and P_phoBR GFP expression.The fold change of P_phoA and P_phoBR GFP expression between 0 and 250 μM of phosphate. For P_phoA, the relative fluorescence intensity of 0 to 250 μM of phosphate is about 2.99 folds. For P_phoBR, it is about 1.88 folds.

The fold change of P_phoA GFP expression is greater than that of P_phoBR between 0 and 250μM of phosphate. Since the GFP expression gradient of P_phoA construct is more significant, it is better to use P_phoA to differentiate various phosphate concentration in the soil.

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.