Phosphate Sensor - phoAp , phoBRp

Potassium sensor

Nitrate sensor (dcuSp)

Introduction

Phosphorus is vital to plant growth and is found in every living plant cell. It is a component of the nucleic acid structure of plants, which regulates protein synthesis. Therefore, it is important in cell division and development of new tissue. It is also associated with complex energy transformations in the plant. Plants deficient in phosphorus are stunted in growth and often have an abnormal dark-green color.

Phosphate sensor Design

phoAp and phoBRp is cross-regulated by phoB and phoR, and is usually repressed under high phosphate concentrations. PhoR behaves as an activator as well as an inactivator for PhoB. When phosphate is limited, PhoR will phosphorylate PhoB and the phosphorylated PhoB will directly activate phoAp and phoBRp. In contrast, when there is phosphate, PhoR will repress PhoB phosphorylation which in turn inactivates the phoAp and phoBRp.

For the constructs design, we have ligated GFP generator to phoAp and phoBRp, respecticely. As a result, under high phosphate concentrations, the green fluorescence intensity will be repressed; while under low phosphate concentrations, green fluorescence will be expressed.

Experiment performed

We have done a characterization on phoAp and phoBRp 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 3 characterization trials.

Result obtained

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Methods

Preparing test medium with different concentrations of phosphate

M9 minimal medium (J. Sambrook & D.W. Russell, 2001) and M9 minimal medium with Tris replacing phosphate were prepared. Test medium with different concentrations of phosphate (0, 10, 30, 50, 100, 150, 200, 250, and 300 µM) were made by mixing the 2 solutions in the following ratio:
60 μl of antibiotics was added in each test medium solution.

Final phosphate concentration (μM)	M9 minimal medium (μl)	M9 minimal medium without phosphate (replaced by Tris) (ml)
0	0.00	60
10	8.58	60
30	25.73	60
50	42.85	60
100	85.71	60
150	130.43	60
200	171.42	60
250	216.26	60
300	260.87	60

phoAp and phoBRp characterization

To characterize phoAp and phoBRp, positive and negative controls were first grown overnight in Luria Broth (LB) medium containing Chloramphenicol at 37^oC. The bacteria were then washed twice with 3 ml M9 minimal medium without phosphate (replaced by Tris), containing Ampicillin. Then, the cells were resuspended in 5 ml M9 minimal medium without phosphate (replaced by Tris) to obtain a final OD₆₀₀ of 4. The cell suspension were then added to the test medium with different concentrations of phosphate (containing Chloramphenicol) in a 96-well deep well plate and further incubated at 37^oC until the OD₆₀₀ of the cells reached the mid-log phase. The fluorescence output was then measured using EnVision multilabel reader.

Team:HKUST-Rice/Phosphate Sensor

Phosphate Sensor - phoAp , phoBRp

Introduction

Phosphate sensor Design

Experiment performed

Result obtained

Methods