Difference between revisions of "Part:BBa K2967017"
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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'' BL21 competent cells. Competent cells were cultured at 37<sup>o<sup>C overnight, and then diluted to OD<sub>600<sub> = 0.4. And then, cultured bacteria at 37<sup>o<sup>C for 1.5 hours, the appropriate concentration of 100μM 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 could effectively activate the expression of the reporter gene. (Fig. 2) | + | For the NO sensor sensitivity testing, we transformed the constructed plasmid with NO sensor into ''E. coli'' BL21 competent cells. Competent cells were cultured at 37<sup>o</sup>C overnight, and then diluted to OD<sub>600</sub> = 0.4. And then, cultured bacteria at 37<sup>o</sup>C for 1.5 hours, the appropriate concentration of 100μM 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 could effectively activate the expression of the reporter gene. (Fig. 2) |
| − | https://static.igem.org/mediawiki/parts/thumb/ | + | https://static.igem.org/mediawiki/parts/thumb/e/eb/T--NEU_China--part--K2967017-2.png/773px-T--NEU_China--part--K2967017-2.png |
| − | '''Figure 2. The response to NO of | + | '''Figure 2. The response to NO of P''yeaR''-luc in ''E. coli'' BL21 competent cells.''' Histogram of Luminescence(RLU): The luminescence signal can be detected from empty vector and PyeaR-luc vector under either SNP induction or not. |
Afterwards, we also transformed the P''yeaR'' based NO sensor in our chassis, ''E. coli Nissle 1917 (EcN)''. The results were consistent with our expectation which confirmed the precise colonization of the engineered ''EcN'' in the inflammatory region (Fig. 3). | Afterwards, we also transformed the P''yeaR'' based NO sensor in our chassis, ''E. coli Nissle 1917 (EcN)''. The results were consistent with our expectation which confirmed the precise colonization of the engineered ''EcN'' in the inflammatory region (Fig. 3). | ||
| − | https://static.igem.org/mediawiki/parts/thumb/ | + | https://static.igem.org/mediawiki/parts/thumb/c/ca/T--NEU_China--part--K2967017-2%2A.png/779px-T--NEU_China--part--K2967017-2%2A.png |
| − | + | ||
| − | + | ||
| − | Luminescence(RLU): The luminescence signal can be detected from empty vector and | + | '''Figure 3. The response to NO of P''yeaR''-luc in ''E. coli Nissle 1917'' .''' Histogram of Luminescence(RLU): The luminescence signal can be detected from empty vector and P''yeaR''-luc vector under either SNP induction or not. |
'''reference''' | '''reference''' | ||
Latest revision as of 12:05, 21 October 2019
Nitrate reporter: PyeaR - Luc composite
The promoter PyeaR 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 PyeaR to inhibit transcription of downstream genes. Then the promoter PyeaR will be activated to express the luciferase.
Usage and Biology
In order to detect the NO specifically and accurately, firstly, we constructed a yeaR based NO sensor in pCDFDuet-1 plasmid, which also used by previous teams’ work. When NO bounds to NsrR (the PyeaR’s transcription inhibitor), halting the repression and allowing the expression of lucifersase. To test the NO sensors’ sensitivity and specificity, we chose luciferase as our reporter gene, because of the extremely sensitivity allows quantification in even small changes in transcription (Fig .1).
Figure 1. Diagram for yeaR based NO sensor system in pCDFDuet-1 plasmid. PyeaR, a promoter which is sensitive to NO. Luciferase, reporter gene. Terminator B0010/B0012, double terminator.
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 cells. Competent cells were cultured at 37oC overnight, and then diluted to OD600 = 0.4. And then, cultured bacteria at 37oC for 1.5 hours, the appropriate concentration of 100μM 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 could effectively activate the expression of the reporter gene. (Fig. 2)
Figure 2. The response to NO of PyeaR-luc in E. coli BL21 competent cells. Histogram of Luminescence(RLU): The luminescence signal can be detected from empty vector and PyeaR-luc vector under either SNP induction or not.
Afterwards, we also transformed the PyeaR based NO sensor in our chassis, E. coli Nissle 1917 (EcN). The results were consistent with our expectation which confirmed the precise colonization of the engineered EcN in the inflammatory region (Fig. 3).
Figure 3. The response to NO of PyeaR-luc in E. coli Nissle 1917 . Histogram of Luminescence(RLU): The luminescence signal can be detected from empty vector and PyeaR-luc vector under either SNP induction or not.
reference
[1] Lin, H. Y., Bledsoe, P. J., & Stewart, V. (2007). Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen-responsive regulator Fnr in Escherichia coli K-12. Journal of bacteriology, 189(21), 7539-7548.
[2] Ljung, T., et al., Rectal nitric oxide assessment in children with Crohn disease and ulcerative colitis. Indicator of ileocaecal and colorectal affection. Scand J Gastroenterol, 2001. 36(10): p. 1073-6.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 193
Illegal NgoMIV site found at 1537
Illegal NgoMIV site found at 1558
Illegal AgeI site found at 1261 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1443