Part:BBa_K216005

PyeaR promoter, responsive to nitrate, nitrite and nitric oxide

PyeaR promoter. This is the promoter of the Escherichia coli yeaR/yoaG operon (see Lin, H.-Y., Bledsoe, P.J., and 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. J. Bacteriol. 189, 7539-7548). Unlike other E. coli promoters responding to nitrate and nitrite, this promoter is not repressed under aerobic conditions.

Usage and Biology

According to Lin et al (2007), this promoter is regulated mainly by phospho-NarL, although phospho-NarP can also activate it if NarL is not present. Repression of the promoter in the absence of nitrate/nitrite is mainly due to the repressor NsrR. Induction is higher under anaerobic conditions than under aerobic conditions, but strong induction still occurs under fully aerobic conditions; this is not true of other known E. coli promoters responsive to nitrate and nitrite. LacZ activities (Miller Units) were as follows:

• anaerobic, complex medium, no induction: 5
• anaerobic, complex medium, 40 mM nitrate: 460
• anaerobic, complex medium, 5 mM nitrite: 97
• anaerobic, minimal medium, no induction: 6
• anaerobic, minimal medium, 40 mM nitrate: 3000
• anaerobic, minimal medium, 5 mM nitrite: 680
• aerobic, minimal medium, no induction: 2
• aerobic, minimal medium, with 40 mM nitrate: 160

NEU_China 2019

The improvement of BBa_K216005

Usage and Biology

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)

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.

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). 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
  

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]