Difference between revisions of "Part:BBa K1682013"
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| − | =P<sub>phoA</sub> | + | =<i>P<sub>phoA</sub></i>- phosphate responsive promoter= |
| − | ===Biology of P<sub>phoA</sub>=== | + | ===Biology of <i>P<sub>phoA</sub></i>=== |
| − | [[File:HKUST-Rice 2015 | + | [[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>.]] |
| − | <i>Escherichia coli</i> (<i>E. coli</i>) detects inorganic phosphate | + | <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>. |
==Constructs for characterization== | ==Constructs for characterization== | ||
| − | [[File:HKUST-Rice 2015 | + | [[File:Team HKUST-Rice 2015 PhoApr.PNG|thumb|500px|center|<b>Fig.2 </b>Phosphate sensing construct with reporter.]] |
With the phosphate (<i>pho</i>) regulon from <i>E. coli</i>, it can be utilized for detecting phosphate level. | With the phosphate (<i>pho</i>) regulon from <i>E. coli</i>, it can be utilized for detecting phosphate level. | ||
| − | To make a phospahte-sensing device, we obtained the promoter, P<sub>phoA</sub>, 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. | + | To make a phospahte-sensing device, we obtained the promoter, <i>P<sub>phoA</sub></i>, 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=== | ===RFU measurement=== | ||
| − | [[File:Team HKUST-Rice 2015 | + | [[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, P<sub>phoA</sub> is induced under phosphate limitation and repressed under high phosphate concentration. The fluorescence intensity dropped by 2.99 folds between 0 to | + | 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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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
| − | <partinfo> | + | <partinfo>BBa_K1682012 SequenceAndFeatures</partinfo> |
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
| − | <partinfo> | + | <partinfo>BBa_K1682012 parameters</partinfo> |
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Revision as of 17:07, 18 September 2015
Contents
PphoA- phosphate responsive promoter
Biology of PphoA
Escherichia coli (E. coli) detects inorganic phosphate from the environment by the PhoR/PhoB two-component system (Hsieh & Wanner, 2010). As illustrated in Figure 1, PphoA 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 PphoA. In contrast, when there is phosphate, PhoR interferes with phosphorylation of PhoB which in turn inactivates PphoA.
Constructs for characterization
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
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 200 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]