Difference between revisions of "Part:BBa K2967026"
 
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<partinfo>BBa_K2967026 short</partinfo>
 
<partinfo>BBa_K2967026 short</partinfo>
  
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'''Usage and Biology'''
  
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This year, we chose BBa_KK2967017 (PyeaR-Luc) <nowiki>https://parts.igem.org/Part:BBa_K2967017</nowiki>
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This year, we chose [https://parts.igem.org/Part:BBa_K2967017, BBa_K2967017]P''yeaR''-Luc as an alternative to our inflammatory sensor, due to its sensitivity to nitrate and nitrite. When nitrate and nitrite enter ''E. coli'', they will be converted to nitric oxide. Then nitric oxide will bind to the repressor protein NsrR that inactivates P''yeaR'' to inhibit transcription of downstream genes.[1]
  
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as an alternative to our inflammatory sensor, due to its sensitivity to nitrate and nitrite. When nitrate and nitrite enter E. coli, they will be converted to nitric oxide. Then nitric oxide will bind to the repressor protein NsrR that inactivates PyeaR to inhibit transcription of downstream genes.[1]
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However, we noticed detectable basal expression (leakage) from the characterization of the NO sensor (P''yeaR''-Luc) (Fig. 2A). To reduce sensor basal background, we inserted an extra NsrR binding sequence (NsrRBS) downstream of P''yeaR'' to create a ‘roadblocking’ effect [2] (Fig. 1).
  
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'''Characterization'''
  
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However, we noticed detectable basal expression (leakage) from the characterization of the most sensitive NO sensor (PyeaR-Luc) (Fig. 2A). To reduce sensor basal background, we integrated two different approaches. For the first approach, we inserted an extra NsrR binding sequence (NsrRBS) downstream of PyeaR to create a ‘roadblocking’ effect [2] (Fig. 1). Compare to the unmodified Pyear-luc system (Fig.2B), the histogram of luminescence data demonstrated that the relative lower luciferase signal in Pyear-NsrRBS system in the absence of NO.
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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.  
  
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__NOTOC__
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For the NO sensor sensitivity testing, we transformed the constructed plasmid with NO sensor into ''E. coli'' BL21 competent cells. 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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https://static.igem.org/mediawiki/parts/thumb/9/95/T--NEU_China--part--ppyear-1.png/800px-T--NEU_China--part--ppyear-1.png.jpeg
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https://static.igem.org/mediawiki/parts/thumb/e/ef/T--NEU_China--part--K2967025K216005-1.png/800px-T--NEU_China--part--K2967025K216005-1.png.jpeg
  
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'''Figure 1. Diagram for NO sensor system in pCDFDuet-1 plasmid.''' PyeaR, a promoter which is sensitive to NO. Native NsrRBS, the native NsrR binding sequence. Extra NsrRBS, the extra NsrR binding sequence. Luciferase, reporter gene. https://static.igem.org/mediawiki/parts/thumb/e/e9/T--NEU_China--part--ppyear-22.png/800px-T--NEU_China--part--ppyear-22.png
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'''Figure 1. Diagram for NO sensor system in pCDFDuet-1.''' P''yeaR'', a promoter which is sensitive to NO. Native NsrRBS, the native NsrR binding sequence. Extra NsrRBS, the extra NsrR binding sequence. Luciferase, reporter gene.  
  
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'''Figure 2. The response to NO sensors. A. The response to NO of Pyear-luc in ECN. Histogram of Luminescence(RLU)''': pcdfduet-1 blank, Pyear-luc without SNP, pcdfduet-1 blank, Pyear-luc with 100μM SNP. '''B. Comparison genetic leakage expression of Pyear-luc and Pyear-NsrRBS-luc systems with or without NO induction.''' Blue bars indicate the luciferase expression percent under the NO induction, while Red bars show the percentage of genetic leakage without NO induction.
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https://static.igem.org/mediawiki/parts/thumb/2/2f/T--NEU_China--part--K2967025K216005-2.png/800px-T--NEU_China--part--K2967025K216005-2.png
  
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The second approach uses protease-based post-translational degradation regulation[2]. First a protein degradation tag (AAV) is added to the reporter protein to reduce the output basal expression. To reduce the background expression without sacrificing the high output, we next incorporated the sensor into a TEV protease-based reporter protein degradation control system (Fig. 3). This hybrid regulation system is sufficient to reduce the sensor’s basal background while also being able to maintain both the sensor’s output amplitude and sensitivity, leading to expanded output dynamic range. However, due to the time limitation, the result is not shown here.
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'''Figure 2. The response to NO sensors.'''
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https://static.igem.org/mediawiki/parts/thumb/0/09/T--NEU_China--part--ppyear-3.png/800px-T--NEU_China--part--ppyear-3.png
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'''A. The response to NO of P''yeaR''-luc in ECN. Histogram of Luminescence(RLU):''' empty vector, P''yeaR''-luc without SNP, empty vector, P''yeaR''-luc with 100μM SNP.
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'''B. Comparison genetic leakage expression of P''yeaR''-luc and P''yeaR''-NsrRBS-luc systems with or without NO induction.''' Blue bars indicated the luciferase expression percent under the NO induction, while Red bars showed the percentage of genetic leakage without NO induction. 100 μM Sodium Nitroprusside Dihydrate (SNP) aqueous solution was used continuously release NO and the final concentration is stable at about 5.5μM,
  
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'''Fig. 3 Tuning the sensor background and output dynamic range via reporter degradation regulation.''' Schematic showing protease-mediated regulation of the background and output dynamic range for an NO sensor. ‘A’ represents the AAV degradation tag. Off state: when there is no NO induction. On state: when there is NO induction.
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'''Conclusion'''
  
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'''reference'''
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Compare to the unmodified P''yeaR''-luc system (Fig.2B), the histogram of luminescence data demonstrated that the relative lower luciferase signal in P''yeaR''-NsrRBS system in the absence of NO.
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'''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.
 
[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.
  
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[2] Merulla, D. & van der Meer, J. R. Regulatable and modulable background expression control in prokaryotic synthetic circuits by auxiliary repressor binding sites. ACS Synth. Biol. 5, 36–45 (2016).<!-- Uncomment this to enable Functional Parameter display
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===Usage and Biology===
 
===Usage and Biology===
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2967026 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2967026 SequenceAndFeatures</partinfo>

Latest revision as of 10:40, 21 October 2019


The yeaR promoter added an extra NsrRBSs-RBS-Luc

Usage and Biology

This year, we chose BBa_K2967017PyeaR-Luc as an alternative to our inflammatory sensor, due to its sensitivity to nitrate and nitrite. When nitrate and nitrite enter E. coli, they will be converted to nitric oxide. Then nitric oxide will bind to the repressor protein NsrR that inactivates PyeaR to inhibit transcription of downstream genes.[1]

However, we noticed detectable basal expression (leakage) from the characterization of the NO sensor (PyeaR-Luc) (Fig. 2A). To reduce sensor basal background, we inserted an extra NsrR binding sequence (NsrRBS) downstream of PyeaR to create a ‘roadblocking’ effect [2] (Fig. 1).

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 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)

800px-T--NEU_China--part--K2967025K216005-1.png.jpeg

Figure 1. Diagram for NO sensor system in pCDFDuet-1. PyeaR, a promoter which is sensitive to NO. Native NsrRBS, the native NsrR binding sequence. Extra NsrRBS, the extra NsrR binding sequence. Luciferase, reporter gene.

800px-T--NEU_China--part--K2967025K216005-2.png

Figure 2. The response to NO sensors. A. The response to NO of PyeaR-luc in ECN. Histogram of Luminescence(RLU): empty vector, PyeaR-luc without SNP, empty vector, PyeaR-luc with 100μM SNP. B. Comparison genetic leakage expression of PyeaR-luc and PyeaR-NsrRBS-luc systems with or without NO induction. Blue bars indicated the luciferase expression percent under the NO induction, while Red bars showed the percentage of genetic leakage without NO induction. 100 μM Sodium Nitroprusside Dihydrate (SNP) aqueous solution was used continuously release NO and the final concentration is stable at about 5.5μM,

Conclusion

Compare to the unmodified PyeaR-luc system (Fig.2B), the histogram of luminescence data demonstrated that the relative lower luciferase signal in PyeaR-NsrRBS system in the absence of NO.

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] Merulla, D. & van der Meer, J. R. Regulatable and modulable background expression control in prokaryotic synthetic circuits by auxiliary repressor binding sites. ACS Synth. Biol. 5, 36–45 (2016).

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 228
    Illegal NgoMIV site found at 1572
    Illegal NgoMIV site found at 1593
    Illegal AgeI site found at 1296
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1478