Difference between revisions of "Part:BBa K1682015"

 
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=P<sub>phoBR</sub>- phosphate responsive promoter=
  
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__NOTOC__
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===Biology of P<sub>phoBR</sub>===
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<partinfo>BBa_K1682015 short</partinfo>
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[[File:HKUST-Rice 2015 Phosphate mechanism.png|thumb|500px|center|<b>Fig.1 </b>Phosphate sensing mechanism of P<sub>phoBR</sub>.]]
  
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<i>Escherichia coli</i> (<i>E. coli</i>) detects inorganic phosphate (P(i)) from the environment by the PhoR/PhoB two-component system (Hsieh & Wanner, 2010). As illustrated in Figure 1, P<sub>phoBR</sub> 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 act as a phospho-donor for the autophosphorylation of PhoB. The phosphorylated PhoB will directly activate P<sub>phoBR</sub>. In contrast, when there is phosphate, PhoR interferes with phosphorylation of PhoB which in turn inactivates P<sub>phoBR</sub>.
  
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==Constructs for characterization==
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[[File:HKUST-Rice 2015 Phosphate c2.PNG|thumb|500px|center|<b>Fig.2 </b>Phosphate sensing construct with reporter.]]
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With the phosphate (<i>pho</i>) regulon from <i>E. coli</i>, it can be utilized for detecting phosphate level.
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To make a phospahte-sensing device, we obtained the promoter, P<sub>phoBR</sub>, and combined it with a GFP reporter, BBa_E0240, in BioBrick RFC10 standard so that the promoter activity in different potassium level can be detected and characterized.
  
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===RFU measurement===
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[[File:HKUST-Rice 2015 Phobr alone.PNG|thumb|500px|center|<b>Fig.3 </b>Activity of P<sub>phoBR</sub> in <i>E. coli</i> DH10B in different phosphate concentrations]]
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As shown in Figure 3, P<sub>phoBR</sub> is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 2.99 folds between 0 to 200μM concentration of phosphate. Furthermore, a plateau is observed starting from the 200 μM phosphate concentration point, suggesting that the dynamic range of P<sub>phoBR</sub> is from 0-200 μM of phosphate.
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1682015 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K1682012 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K1682015 parameters</partinfo>
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<partinfo>BBa_K1682012 parameters</partinfo>
 
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Revision as of 20:33, 13 September 2015

PphoBR- phosphate responsive promoter

Biology of PphoBR

Fig.1 Phosphate sensing mechanism of PphoBR.

Escherichia coli (E. coli) detects inorganic phosphate (P(i)) from the environment by the PhoR/PhoB two-component system (Hsieh & Wanner, 2010). As illustrated in Figure 1, PphoBR 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 act as a phospho-donor for the autophosphorylation of PhoB. The phosphorylated PhoB will directly activate PphoBR. In contrast, when there is phosphate, PhoR interferes with phosphorylation of PhoB which in turn inactivates PphoBR.

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, PphoBR, and combined it with a GFP reporter, BBa_E0240, in BioBrick RFC10 standard so that the promoter activity in different potassium level can be detected and characterized.

RFU measurement

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

As shown in Figure 3, PphoBR is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 2.99 folds between 0 to 200μM concentration of phosphate. Furthermore, a plateau is observed starting from the 200 μM phosphate concentration point, suggesting that the dynamic range of PphoBR 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
    COMPATIBLE WITH RFC[1000]