Team:HKUST-Rice/Phosphate Sensor
Phosphate Sensor
Introduction
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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 phoAppromoter.
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) |
| Eve | Jackson | 94 | |
| John | Doe | 80 |
Result obtained
Lorem ipsum dolor sit amet, pro aeque temporibus eu, eum qualisque assueverit te. Ad est admodum epicuri suscipit, te alterum aliquando adversarium usu, pro ex omnesque luptatum comprehensam. In vix alia percipit gloriatur, no ferri lorem aliquando cum. Fugit concludaturque sed ne, ea sumo dico adolescens eos, quo eu pertinax expetendis. An his omnes instructior, vide possim eam id. Te cum enim sale offendit, vocent copiosae luptatum ut per. Eam in alienum accusamus, et probo reque vix. Vivendum necessitatibus qui ad, no vis enim veniam perpetua. Eu pri habemus senserit, dicit tation expetenda usu et. Sea eu dolor deserunt dissentias, sed an oportere moderatius assueverit. Usu te tation gloriatur, vidit tollit utinam mea id.
Lorem ipsum dolor sit amet, pro aeque temporibus eu, eum qualisque assueverit te. Ad est admodum epicuri suscipit, te alterum aliquando adversarium usu, pro ex omnesque luptatum comprehensam. In vix alia percipit gloriatur, no ferri lorem aliquando cum. Fugit concludaturque sed ne, ea sumo dico adolescens eos, quo eu pertinax expetendis. An his omnes instructior, vide possim eam id. Te cum enim sale offendit, vocent copiosae luptatum ut per. Eam in alienum accusamus, et probo reque vix. Vivendum necessitatibus qui ad, no vis enim veniam perpetua. Eu pri habemus senserit, dicit tation expetenda usu et. Sea eu dolor deserunt dissentias, sed an oportere moderatius assueverit. Usu te tation gloriatur, vidit tollit utinam mea id.