Re-characterization: of Phosphate Sensors in brief

• Phosphate associate with essential growth factors for plant root development as well as seed production.
• Our re-characterization of P_phoA , P_phoBR data provide dynamic range of these promoters.

Phosphate Sensor Mechanism

Escherichia coli detects inorganic phosphate 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 acts as a phospho-donor for the phosphorylation of PhoB. The phosphorylated PhoB will directly activate P_phoA and P_phoBR. In contrast, when there is high phosphate concentration, PhoR interferes with phosphorylation of PhoB which in turn inactivates both P_phoA and P_phoBR.

Figure 1. Phosphate sensing machine of P_phoA and P_phoBR

Phosphate Sensor Design

Figure 2. Phosphate sensor constructs. (a) P_phoA with GFP generator and (b) P_phoBR with GFP generator.

Native P_phoA and P_phoBR from E. coli can be utilized as our phosphate promoter. They were cloned and ligated with the GFP generator (pSB1C3-BBa_I13504) respectively. GFP sign is used as a promoter activity readout under different phosphate concentrations.

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.

Please visit Phosphate sensor Experiment Protocol for more details.

Results

After measuring the GFP signal intensity using an EnVision® multilabel reader, the fluorescence signals were normalized against the cell density.

Figure 3. Characterization of P_phoA and P_phoBR in M9 minimal medium. GFP expression of pSB1C3-P_phoA-BBa_I13504 and pSB1C3-P_phoBR-BBa_I13504 in 0, 10, 30, 50, 100, 150, 200, 250 and 300 µM phosphate concentration are shown. Error bars were presented in SEM. .

As shown in Figure 3a, P_phoA is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 2.99 folds between 0 to 200 μM 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 3b, 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 250 μM of phosphate concentration point, suggesting that the dynamic range of P_phoBR is from 0-250 μM of phosphate.

Discussion

To select the promoter with better resolution of different phosphate concentrations, we compared the activity difference of two promoters between 0 and 250 μM of phosphate.

Figure 4. Comparison between 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 fluorescence intensity from 0 to 250 μM of phosphate dropped by 2.99 folds. For P_phoBR, it dropped by 1.88 folds. Error bars were presented in SEM.

The fold change of P_phoA GFP expression is greater than that of P_phoBR between 0 and 250μM of phosphate. P_phoA is suggested to be used if a better resolution of phosphate concentration is desired.

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.