Difference between revisions of "Part:BBa K2967023"
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In order to simulate the inflammatory NO, 100 μM Sodium Nitroprusside Dihydrate (SNP) aqueous solution was used to continuously release NO and the final concentration was stable at about 5.5μM, which was the same as the NO concentration in IBD patients [1]. We used 100 μM SNP solutions for NO sensor sensitivity testing. | In order to simulate the inflammatory NO, 100 μM Sodium Nitroprusside Dihydrate (SNP) aqueous solution was used to continuously release NO and the final concentration was stable at about 5.5μM, which was the same as the NO concentration in IBD patients [1]. We used 100 μM SNP solutions for NO sensor sensitivity testing. | ||
| − | For the NO sensor sensitivity testing, we transformed the constructed plasmid with NO sensor into ''E. coli'' | + | For the NO sensor sensitivity testing, we transformed the constructed plasmid with NO sensor into ''E. coli BL21'' competent cell. Competent cells are cultured at 37 ℃ overnight, and then diluted to OD<sub>600</sub> = 0.4. And then, culture bacteria at 37 ℃ for 1.5 hours, the appropriate concentration of inducer SNP aqueous solution were added. After 2 hours of SNP induction, we detected the expression of the luciferase by Luciferase assay (from Beyotime RG005). The Luminescence data indicated that the NO released by the SNP aqueous solution can effectively activate the expression of the reporter gene. (Fig. 2) |
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| + | '''Figure 2. Design and characterization of the ''ytfE'' based NO sensors. A. Diagram for ''ytfE'' based NO sensor system in pCDFDuet-1 plasmid.''' P''ytfE'', a promoter which is sensitive to NO. Luciferase, reporter gene. Terminator B0010/B0012, double terminator. '''B. Comparison genetic expression of P''yeaR''-Luc and P''year''--Luc systems with or without NO induction.''' Red bars indicate the P''ytfE''’s luciferase expression percent under the NO induction. Blue bars indicate the P''yeaR''’s luciferase expression percent under the NO induction. Yellow bars show the percentage of P''year''’s leakage without NO induction. | ||
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'''Conclusion''' | '''Conclusion''' | ||
Revision as of 07:06, 19 October 2019
PytfE promoter, responsive to nitrate, nitrite and nitric oxide
The promoter PytfE is sensitive to nitrate and nitrite. When nitrate and nitrite enter E. coli, they are converted to nitric oxide. Nitric oxide binds to the repressor protein NsrR, which inactivates PytfE to inhibit transcription of downstream genes. Then the promoter PytfE will be activated to express the luciferase.
Usage and Biology
The ytfE is induced by NO as part of the NsrR regulon.ytfE promoter (PytfE) are normally repressed by NsrR, a protein native to most E. coli cells. When nitrate or nitric oxide is bound to NsrR, halting the repression and allowing the transcription of downstream genes. Based on Saraiva’s team’s work, the ytfEΔ strain is much more sensitive to nitric oxide than the parental E. coli strain [1]. Thus, we constructed a novel sensing system PytfE-Luc to compare with our previous sensors to demonstrate the most sensitive and specific NO sensor.
Characterization
In order to simulate the inflammatory NO, 100 μM Sodium Nitroprusside Dihydrate (SNP) aqueous solution was used to continuously release NO and the final concentration was stable at about 5.5μM, which was the same as the NO concentration in IBD patients [1]. We used 100 μM SNP solutions for NO sensor sensitivity testing.
For the NO sensor sensitivity testing, we transformed the constructed plasmid with NO sensor into E. coli BL21 competent cell. Competent cells are cultured at 37 ℃ overnight, and then diluted to OD600 = 0.4. And then, culture bacteria at 37 ℃ for 1.5 hours, the appropriate concentration of inducer SNP aqueous solution were added. After 2 hours of SNP induction, we detected the expression of the luciferase by Luciferase assay (from Beyotime RG005). The Luminescence data indicated that the NO released by the SNP aqueous solution can effectively activate the expression of the reporter gene. (Fig. 2)
Figure 2. Design and characterization of the ytfE based NO sensors. A. Diagram for ytfE based NO sensor system in pCDFDuet-1 plasmid. PytfE, a promoter which is sensitive to NO. Luciferase, reporter gene. Terminator B0010/B0012, double terminator. B. Comparison genetic expression of PyeaR-Luc and Pyear--Luc systems with or without NO induction. Red bars indicate the PytfE’s luciferase expression percent under the NO induction. Blue bars indicate the PyeaR’s luciferase expression percent under the NO induction. Yellow bars show the percentage of Pyear’s leakage without NO induction.
Conclusion
In conclusion, the results demonstrate that the ytfE based NO sensor’s output is nearly five times higher than the yeaR based NO sensor’s (Fig. 2B), which convinced us that the novel NO sensor, PytfE is more sensitive.
reference
[1]Justino, M.C., et al., Escherichia coli YtfE is a di-iron protein with an important function in assembly of iron-sulphur clusters. FEMS Microbiol Lett, 2006. 257(2): p. 278-84.
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]