Difference between revisions of "Part:BBa K1682012"

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==Design==
 
==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The inducible promoter BBa_K1682012, created by 2015 HKUST-Rice iGEM, was originally made to control the expression of the PhoA alkaline phosphatase gene following activation by the phosphorylated PhoB transcription factor.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.
 
The inducible promoter BBa_K1682012, created by 2015 HKUST-Rice iGEM, was originally made to control the expression of the PhoA alkaline phosphatase gene following activation by the phosphorylated PhoB transcription factor.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.
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==EXPERIENCE (Lambert_GA 2021)==
 
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Utilizing the modified phosphate range and characterization protocol (See: [https://2021.igem.org/Team:Lambert_GA/Pho#:~:text=Engineering%20Success%20page.-,Characterization%20Protocol,-The%20failed%20characterization Modified Protocol]), we characterized the BBa_K3725110 phosphate sensor. However, the characterization data (Figure 1) yielded insignificant levels of fluorescence, thus leading us to conclude that the promoter is not sensitive enough to extracellular phosphate levels.
 
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[[File:ASDJFCJBASJHBFD.png|thumb|center|800px|Figure 2. Characterization curve for BBa_K3725110 for phosphate concentrations between 0μM and 100μM.  ]]
 
  
 
==Constructs for characterization==
 
==Constructs for characterization==

Revision as of 19:55, 17 December 2021

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]