Difference between revisions of "Part:BBa K1682013"

(→‎PphoA - I13504 phosphate responsive promoter with GFP generator)
(→‎PphoA - I13504 phosphate responsive promoter with GFP generator)
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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>Escherichia coli</i> (<i>E. coli</i>) detects inorganic phosphate from the environment by the PhoR/PhoB two-component system (Hsieh & Wanner, 2010). As illustrated in Figure 1, <i>P<sub>phoA</sub></i> 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 acts as a phospho-donor for the autophosphorylation of PhoB. The phosphorylated PhoB will directly activate <i>P<sub>phoA</sub></i>. In contrast, when there is phosphate, PhoR interferes with phosphorylation of PhoB which in turn inactivates <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 examples is <i>phoA</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>.
  
 
==Constructs for characterization==
 
==Constructs for characterization==
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===RFU measurement===
 
===RFU measurement===
 
[[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]]
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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 200 μM concentration of phosphate. Furthermore, a plateau is observed starting from the 200 μM phosphate concentration point, suggesting that the dynamic range of <i>P<sub>phoA</sub></i> is from 0-200 μM of phosphate.
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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 200 μM phosphate concentration point, suggesting that the dynamic range of <i>P<sub>phoA</sub></i> is from 0-200 μM of phosphate.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 02:50, 19 September 2015

PphoA - I13504 phosphate responsive promoter with GFP generator

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, one of the examples is phoA. 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.

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 200 μM phosphate concentration point, suggesting that the dynamic range of PphoA is from 0-200 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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 755