Part:BBa_K3527003
PhoA-signal peptide-EGFP
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
PhoA-signal peptide-EGFP
In E. coli, the expression level of PhoA gene is affected by the concentration of phosphate. BBa_K3527003 uses enhancer green fluorescence protein (EGFP) gene as the reporter gene to construct the fusion detection vector PhoA-EGFP, and then the sythetic generator can be used as the biosensor displays different fluorescence intensities according to the level of phosphorus content in the water, which can easily and quickly detect the content of phosphorus in different water bodies
Contribution
BBa K3527003 is a composite part constituting of enhanced green fluorescent protein gene under control of phoA promoter (basic parts BBa_K3527000). The similar design was seen for composite parts BBa_K1139201 and BBa_K737024. The part BBa_K737024 does not have experimental data showing its function and the part BBa_K1139201 has sufficient data showing its function but no practical application.
Engineering success
Fluorescence intensity of enhanced green fluorescent protein (EGFP) regulated by phosphate concentration Bacteria containing phoA-EGFP showed that significant fluorescence intensity difference compared to the control in the absence of phosphate in the medium (Figure 2). When the phosphate concentration added into the culture, the fluorescence intensity decreased to a level similar to control culture. In the case of phosphorus deficiency (0-0.2mM), the detectable fluorescence of phoA-EGFP was the most significant difference compared with the control group.
Figure 2. Different concentrations of phosphate were added to the culture medium for treatment, within 15 minutes, compared with the control group, the bacteria containing PhoA-GFP plasmid had a higher detectable fluorescence value in the 0-0.2mM concentration range.
The experiment results above demonstrate the functionality of our phoA-EGFP (BBa_K3527003) in detecting phosphate concentration.
Determination the best of phosphate detection time for phoA-EGFP system As shown in Figure 3, with the extending of culturing time, the GFP fluorescence signal intensity decreased. This suggests that phoA-EGFP can detect phosphorus deficiency signals in a short period of time (approximately within 1 h). It has great application potential in detection of water environment polluted by phosphate.
Figure 3. Fluorescence intensity of phoA-EGFP system changes with culturing time.
The experiment results above also indicate that the functionality of our phoA-EGFP (BBa_K3527003) in determining the best detection time of phosphate.
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