Team:HKUST-Rice/Phosphate Sensor
Phosphate Sensor - phoAp , phoBRp
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 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, phoAp and phoBRp 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 phoAp and phoBRp. In contrast, when there is phosphate, PhoR interferes with phosphorylation of PhoB which in turn inactivates both phoAp and phoBR.
With the phosphate (Pho) regulon from E. coli,it can be utilized for detecting phosphate level. phoAp and 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 .
Figure 2. Characterization of phoAp and phoBRp in M9 minimal medium. GFP expression of pSB1C3-phoAp-BBa_I13504 and pSB1C3-phoBRp-BBa_I13504 in 0,10,30,50,100,150,200 and 300 µM phosphate concentration are shown. .
As shown in Figure 2a, phoAp 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 200μM phosphate concentration point, suggesting that the dynamic range of phoAp is from 0-200μM of phosphate.
As shown in Figure 2b, phoBRp is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 1.88 folds between 0 to 250μM concentration of phosphate. Furthermore, a plateau is observed starting from the 250μM of phosphate concentration point, suggesting that the dynamic range of phoBRp is from 0-250μM of phosphate.
Discussion
In order to select a better phosphate sensor, the fold change of phoAp and phoBRp were being compared.
Figure 3. Fold change of phoAp and phoBRp GFP expression.The fold change of phoAp and phoBRp GFP expression between 0 and 250μM of phosphate. For phoAp, the relative fluorescence intensity of 0 to 250μM of phosphate is about 2.99 folds. For phoBRp, it is about 1.88 folds.
The fold change of phoAp GFP expression is greater than that of phoBRp between 0 and 250μM of phosphate. Since the GFP expression gradient of phoAp construct is more significant, it is better to use phoAp 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.