Revision as of 03:28, 30 August 2015

Phosphate Sensor - phoAp

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. Phosphorus is, therefore, 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 promoter (Hsieh, Y. J., & Wanner, B. L.,2010).is cross-regulated by phoB and phoR, and is usually repressed under high phosphate concentration. 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 the expression of phoAp promoter. In contrast, when there is phosphate, phoR will repress PhoB phosphorylation which in turns inactivates phoAp promoter.

For the constructs design, we have ligated GFP generator to the phoAp promoter. As a result, when under high phosphate concentration, the green fluorescence intensity will be repressed, while under low phosphate concentration, the situation will be vice versa.

Experiment that we did

We have done a characterization on pSB1C3-phoAp-BBa_I13504 using Luria broth (LB) medium. Quantitative characterization on the promoter was done by measuring the fluorescence signal intensity using an EnVision multilabel reader.

E. coli strain DH10B was used, and the concentration of the characterization of phoAp promoter was from 0 to 300 µM phosphate, with an intervals of 50µM.

Preparing test medium with different concentration of phosphate

We have prepared a solution of M9 minimal medium (J. Sambrook & D.W. Russell, 2001) and a solution of M9 minimal medium with Tris replacing phosphate. Test medium with different concentration of phosphate (0, 10, 30, 50, 100, 150, 200, 250, 300 µM) were made by mixing the 2 solution in the following ratio.

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

phoAp promoter Characterization

pSB1C3-phoAp-BBa_I13504, positive control and negative control were first grown overnight in 5 ml 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 5ml M9 minimal medium without phosphate(replaced by Tris) to obtain a final OD₆₀₀ of 4. 50 μl of the prepared cell suspension were then added into 950μl of test medium with different concentrations of phosphate (containing Chloramphenicol) in the 96-well deep well plate and further incubate at 37^oC until the OD₆₀₀ of the cells reaches the mid-log phase. The fluorescence output were then measured using EnVision multilabel reader.

This result was obtained by combining 3 characterization trials.

Result obtained

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