Difference between revisions of "Part:BBa K2967025"
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<partinfo>BBa_K2967025 short</partinfo>
 
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Improvement of Pyear
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'''Usage and Biology'''
  
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This year, we chose BBa_K    (PyeaR-Luc) as an alternative to our inflammatory sensor, due to promoter PyeaR is sensitive 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 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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===Usage and Biology===
 
===Usage and Biology===
  
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<!-- --><span class="h3bb">Sequence and Features</span>
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<span class="h3bb">Sequence and Features</span>
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<partinfo>BBa_K2967025 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2967025 SequenceAndFeatures</partinfo>
  
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This year, we chose BBa_K2967017 (PyeaR-Luc, https://parts.igem.org/Part:BBa_K2967017) 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 (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).
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'''Characterization'''
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In order to simulate the inflammatory NO, 100 μM Sodium Nitroprusside Dihydrate (SNP) aqueous solution was used continuously release NO and the final concentration is stable at about 5.5μM, which is the same as the NO concentration in IBD patients . We used 100 μM SNP solutions for NO sensor sensitivity testing.
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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 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.
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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. 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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'''Conclusion'''
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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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'''reference'''
  
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[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  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2967025 parameters</partinfo>
 
<partinfo>BBa_K2967025 parameters</partinfo>
 
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Revision as of 15:17, 7 October 2019


The yeaR promoter added an extra NsrR Binding Sequences

Usage and Biology


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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

This year, we chose BBa_K2967017 (PyeaR-Luc, https://parts.igem.org/Part:BBa_K2967017) 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 continuously release NO and the final concentration is stable at about 5.5μM, which is the same as the NO concentration in IBD patients . 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)

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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.

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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. 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).