Difference between revisions of "Part:BBa K1682012"
| Line 4: | Line 4: | ||
===Biology of <i>P<sub>phoA</sub></i>=== | ===Biology of <i>P<sub>phoA</sub></i>=== | ||
| − | [[File:Team HKUST-Rice 2015 Phosmech pr.PNG|thumb|500px|center|<b> | + | [[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>.]] |
<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>. | <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>. | ||
| Line 14: | Line 14: | ||
Utilizing the modified phosphate range and characterization protocol (See: Modified Protocol under Pho), 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. | Utilizing the modified phosphate range and characterization protocol (See: Modified Protocol under Pho), 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. | ||
| − | [[File:ASDJFCJBASJHBFD.png|thumb|center|800px|Figure | + | [[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== | ||
| − | [[File:Team HKUST-Rice 2015 PhoApr.PNG|thumb|500px|center|<b> | + | [[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. | ||
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. | 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=== | ||
| − | [[File:Team HKUST-Rice 2015 Phoaa.gif|thumb|500px|center|<b> | + | [[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]] |
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. | 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. | ||
Revision as of 22:09, 21 October 2021
PphoA promoter
PphoA- phosphate responsive promoter
Contents
Biology 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.
EXPERIENCE
Utilizing the modified phosphate range and characterization protocol (See: Modified Protocol under Pho), 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.
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 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]