Difference between revisions of "Part:BBa K1682012"
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=P<sub>phoA</sub>- phosphate responsive promoter=
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<partinfo>BBa_K1682012 short</partinfo>
  
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===Biology of P<sub>phoA</sub>===
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<i>P<sub>phoA</sub></i>- phosphate responsive promoter
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[[File:HKUST-Rice 2015 Phosphate mechanism.png|thumb|500px|center|<b>Fig.1 </b>Phosphate sensing mechanism of P<sub>phoA</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>phoA</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>phoA</sub>. In contrast, when there is phosphate, PhoR interferes with phosphorylation of PhoB which in turn inactivates P<sub>phoA</sub>.
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===Biology of <i>P<sub>phoA</sub></i>===
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[[File:Team HKUST-Rice 2015 Phosmech pr.PNG|thumb|500px|center|<b>Figure 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, 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>.
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==Design==
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The inducible promoter BBa_K1682012, created by 2015 HKUST-Rice iGEM, was originally made to control the expression of the PhoA alkaline phosphatase gene following activation by the phosphorylated PhoB transcription factor. By replacing the downstream PhoA gene with GFP,  the part expresses fluorescence in the presence of low levels of extracellular phosphate.
  
 
==Constructs for characterization==
 
==Constructs for characterization==
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[[File:HKUST-Rice 2015 Phosphate c1.PNG|thumb|500px|center|<b>Fig.2 </b>Phosphate sensing construct with reporter.]]
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[[File:Team HKUST-Rice 2015 PhoApr.PNG|thumb|500px|center|<b>Figure 3. </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.  
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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 potassium level can be detected and characterized.
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To make a phospahte-sensing device, we obtained the promoter, <i>P<sub>phoA</sub></i>, and combined it with a GFP reporter, <partinfo>BBa_E0240</partinfo>, in BioBrick RFC10 standard so that the promoter activity in different phosphate level can be detected and characterized.
  
 
===RFU measurement===
 
===RFU measurement===
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[[File:Team HKUST-Rice 2015 phoa.PNG|thumb|500px|center|<b>Fig.3 </b>Activity of P<sub>phoA</sub> in <i>E. coli</i> DH10B in different phosphate concentrations]]
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[[File:Team HKUST-Rice 2015 Phoaa.gif|thumb|500px|center|<b>Figure 4. </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, 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 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>phoA</sub> 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 300 μM phosphate concentration point, suggesting that the dynamic range of <i>P<sub>phoA</sub></i> is from 0-300 μM of phosphate.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
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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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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K1682012 parameters</partinfo>
 
<partinfo>BBa_K1682012 parameters</partinfo>
 
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= Prairie_iGEM2022_UManitoba =
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====Summary====
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The 2022 UManitoba iGEM team uses this part, along with [https://parts.igem.org/Part:BBa_B0032 BBa_B0032], [https://parts.igem.org/Part:BBa_K3033009 BBa_K3033009], and [https://parts.igem.org/Part:BBa_B0015 BBa_B0015] to create a sensor to detect presence of phosphate based on fluorescence of EGFP ([https://parts.igem.org/Part:BBa_K3033009 BBa_K3033009]). Different concentrations of phosphate are tested to identify a suitable concentration range. The sensor is cloned into three different backbones (pUCIDT and pSB1C3) to optimize EGFP expression.
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====Characterization in pUCIDT with different concentrations of phosphate====
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BL21(DE3) cells transformed with a phosphate sensor on pUCIDT backbone are used for testing.
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Expression in Erlenmeyer flask with 0 uM and 500 uM sodium phosphate. For data below, normalized fluorescence intensity according to an optical density at 600 nm (OD600) are reported.
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[[File:Phosphate sensor.png|600px|center|Phosphate sensor]]
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'''Figure 1. Expression of EGFP detected in phosphate deficiency condition.'''
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[[File:Phosphate Hill.png|600px|center|Phosphate Hill]]
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'''Figure 2. The fitted curve of EGFP expression in response to phosphate for PhoA promoter.'''
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====Conclusion====
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From our result, we can conclude that this phosphate sensor part can be used to detect phosphate at below 100 uM phosphate when this part is used with a high copy number plasmid such as pUCIDT.

Latest revision as of 05:18, 7 October 2022

PphoA promoter

PphoA- phosphate responsive promoter

Biology of PphoA

Figure 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.

Design

The inducible promoter BBa_K1682012, created by 2015 HKUST-Rice iGEM, was originally made to control the expression of the PhoA alkaline phosphatase gene following activation by the phosphorylated PhoB transcription factor. By replacing the downstream PhoA gene with GFP, the part expresses fluorescence in the presence of low levels of extracellular phosphate.

Constructs for characterization

Figure 3. 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

Figure 4. 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]

Prairie_iGEM2022_UManitoba

Summary

The 2022 UManitoba iGEM team uses this part, along with BBa_B0032, BBa_K3033009, and BBa_B0015 to create a sensor to detect presence of phosphate based on fluorescence of EGFP (BBa_K3033009). Different concentrations of phosphate are tested to identify a suitable concentration range. The sensor is cloned into three different backbones (pUCIDT and pSB1C3) to optimize EGFP expression.

Characterization in pUCIDT with different concentrations of phosphate

BL21(DE3) cells transformed with a phosphate sensor on pUCIDT backbone are used for testing. Expression in Erlenmeyer flask with 0 uM and 500 uM sodium phosphate. For data below, normalized fluorescence intensity according to an optical density at 600 nm (OD600) are reported.

Phosphate sensor

Figure 1. Expression of EGFP detected in phosphate deficiency condition.

Phosphate Hill

Figure 2. The fitted curve of EGFP expression in response to phosphate for PhoA promoter.

Conclusion

From our result, we can conclude that this phosphate sensor part can be used to detect phosphate at below 100 uM phosphate when this part is used with a high copy number plasmid such as pUCIDT.