Difference between revisions of "Part:BBa K1682012"

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[[File:Team HKUST-Rice 2015 Phosmech pr.PNG|thumb|500px|center|<b>Fig.1 </b>Phosphate sensing mechanism of <i>P<sub>phoA</sub></i>.]]
 
[[File:Team HKUST-Rice 2015 Phosmech pr.PNG|thumb|500px|center|<b>Fig.1 </b>Phosphate sensing mechanism of <i>P<sub>phoA</sub></i>.]]
  
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<i>E. coli</i> has multiple native phosphorus sensing and regulation systems that we could use in the construct. Among them, we chose the PhoR/PhoB two-component system (TCS). It contains a sensory histidine kinase PhoR and a partner DNA-binding response regulator PhoB. PhoR is activated under low phosphate concentration, which will then phosphorylate PhoB. The phospho-PhoB is then capable of activating expression of the Pho regulon genes, one of the two examples are <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i> . In high phosphate concentration, <i>phoR</i> is turned into an inhibitory state, which interferes with phosphorylation of PhoB. PhoB is, thus, not capable of activating expression of <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i>.
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<i>E. coli</i> has multiple native phosphorus sensing and regulation systems that we could use in the construct. Among them, we chose the PhoR/PhoB two-component system (TCS). It contains a sensory histidine kinase PhoR and a partner DNA-binding response regulator PhoB. PhoR is activated under low phosphate concentration, which will then phosphorylate PhoB. The phospho-PhoB is then capable of activating expression of the Pho regulon genes, two of the examples are <i>phoA</i> and <i>phoBR</i>. In high phosphate concentration, <i>phoR</i> is turned into an inhibitory state, which interferes with phosphorylation of PhoB. PhoB is, thus, not capable of activating expression of <i>phoA</i> and <i>phoBR</i>.
  
 
==Constructs for characterization==
 
==Constructs for characterization==
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[[File:Team HKUST-Rice 2015 Phoaa.gif|thumb|500px|center|<b>Fig.3 </b>Activity of <i>P<sub>phoA</sub></i> in <i>E. coli</i> DH10B in different phosphate concentrations]]
 
[[File:Team HKUST-Rice 2015 Phoaa.gif|thumb|500px|center|<b>Fig.3 </b>Activity of <i>P<sub>phoA</sub></i> in <i>E. coli</i> DH10B in different phosphate concentrations]]
 
As shown in Figure 3, <i>P<sub>phoA</sub></i> is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 2.99 folds between 0 to 300 μM concentration of phosphate. Furthermore, a plateau is observed starting from the 300 μM phosphate concentration point, suggesting that the dynamic range of <i>P<sub>phoA</sub></i> is from 0-300 μM of phosphate.
 
As shown in Figure 3, <i>P<sub>phoA</sub></i> is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 2.99 folds between 0 to 300 μM concentration of phosphate. Furthermore, a plateau is observed starting from the 300 μM phosphate concentration point, suggesting that the dynamic range of <i>P<sub>phoA</sub></i> is from 0-300 μM of phosphate.
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===References===
 
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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.
 
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1682012 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1682012 SequenceAndFeatures</partinfo>
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<br><br>
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===References===
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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.
  
  

Revision as of 02:59, 19 September 2015

PphoA- phosphate responsive promoter

Biology of PphoA

Fig.1 Phosphate sensing mechanism of PphoA.

E. coli has multiple native phosphorus sensing and regulation systems that we could use in the construct. Among them, we chose the PhoR/PhoB two-component system (TCS). It contains a sensory histidine kinase PhoR and a partner DNA-binding response regulator PhoB. PhoR is activated under low phosphate concentration, which will then phosphorylate PhoB. The phospho-PhoB is then capable of activating expression of the Pho regulon genes, two of the examples are phoA and phoBR. In high phosphate concentration, phoR is turned into an inhibitory state, which interferes with phosphorylation of PhoB. PhoB is, thus, not capable of activating expression of phoA and phoBR.

Constructs for characterization

Fig.2 Phosphate sensing construct with reporter.

With the phosphate (pho) regulon from E. coli, it can be utilized for detecting phosphate level. To make a phospahte-sensing device, we obtained the promoter, PphoA, and combined it with a GFP reporter, BBa_E0240, in BioBrick RFC10 standard so that the promoter activity in different phosphate level can be detected and characterized.

RFU measurement

Fig.3 Activity of PphoA in E. coli DH10B in different phosphate concentrations

As shown in Figure 3, PphoA is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 2.99 folds between 0 to 300 μM concentration of phosphate. Furthermore, a plateau is observed starting from the 300 μM phosphate concentration point, suggesting that the dynamic range of PphoA is from 0-300 μM of phosphate.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



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.