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

SEOUL-KOREA_2023

pAmpR_RBS-nsrR-PyeaR-RBS_mScarlet_I3

Usage and Biology

Figure 1. Optimized nsrR expression system and nitrate induced reporter system

Our team's nitrate sensor (""BBa_K4948031"") was more sophisticated, based on the PyeaR promoter (""BBa_K216005"") from Edinburgh iGEM 2009. Since nsrR is a nitrate-dependent transcriptional repressor, a high amount of nsrR requires a high concentration of nitrate, resulting in a low performance sensor. Therefore, it is essential to determine the optimal concentration of nsrR. To this end, we used different constitutive expression units to control the intracellular concentration of nsrR. In addition, we added the hmpA1 site, which is known as the binding site of nsrR, between the yeaR promoter and the ribosome binding site.

Figure 2. Measuring the strength of a promoter with the reporter system

Figure 3. Changes in fluorescence intensity depending on the strength of the ribosome binding site in nitrate sensing

Our sensor is capable of detecting trace amounts of nitrate than existing sensors (Figure 4). To increase the intensity of nitrate-induced fluorescence, we added a strong RBS between the yeaR promoter and reporter gene, and in addition to this, we used a medium with higher nutrients to compensate for nitrate-induced cytotoxicity.

Figure 4. Fluorescence intensity by nitrate concentration

NEU_China 2019

The yeaR promoter added an extra NsrR Binding Sequences

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)

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.

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.

MIT_MAHE 2020

Summary

This is the promoter of the Escherichia coli yeaR/yoaG operon; responsive to nitrate, nitrite and nitric oxide.This 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. Unlike other E. coli promoters responding to nitrate and nitrite, this promoter is not repressed under aerobic conditions.

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

2021 XHD-Wuhan-A-Chian

Usage and Biology

Introduction

We improved part: BBa_K216005 (PyeaR promoter), which is the promoter of the Escherichia coli yeaR/yoaG operon. The most remarkable feature of this promoter is its ability to sense nitrate and nitrite. In order to better regulate the response of the promoter to nitrate, we use machine learning models to predict and design new PyeaR sequences. Compared to the original sequence, five or six bases have been changed.

Construction of improved PyeaR

Based on the original sequence, we designed and predicted three mutation sequences that can increase the intensity of the promoter by using our machine learning model. By modifying the PCR primers, we successfully obtained the three mutated PyeaR promoters. Through homologous recombination, we replaced the wild-type promoter with the improved promoter.

Characterization

We used machine learning methods to predict the promoter strength after mutation, and the results are shown in Figure 1.

Figure 1 The predicted strength of the wild-type PyeaR and mutant PyeaR
In order to verify the true strength of our redesigned promoter, we replaced the wild-type promoter with a mutant promoter. After culturing the engineered bacteria overnight at 220 rpm, it was reactivated at a ratio of 1:100 in LB liquid medium for 4 hours. And then we tested OD₅₈₈ of the samples every half hour for 6 hours. The results are shown in Figure 2 and Figure 3. The results show that mutant PyeaR (BBa_K3926002) has a stronger promoter strength than the wild-type.

Figure 2 Verified the strength of the wild-type PyeaR and mutant PyeaR


Figure 3 The expression levels of amilCP genes are different between wild-type PyeaR and mutant PyeaR

Conclusion

Compare to the unmodified PyeaR promoter (BBa_K216005, WT-PyeaR in Figure 3), the histogram of amilCP (OD₅₈₈) data demonstrated that the higher strength in mutant PyeaR. In short, we have successfully improved the pyear promoter to make it have a higher promoter strength.

Prairie_iGEM2022_UManitoba

Summary

The 2022 UManitoba iGEM team uses this part, along with BBa_B0032, BBa_K3033009, and BBa_B0015 to create a sensor to detect the presence of nitrate based on fluorescence of EGFP (BBa_K3033009). Different concentrations of nitrate are tested to identify a suitable concentration range. The sensor is cloned into three different backbones (pUCIDT, pSB1C3, and pET28b) to optimize EGFP expression.

Characterization in pUCIDT with different concentrations of nitrate

BL21(DE3) cells transformed with nitrate sensor on pUCIDT backbone are used for testing. Expression in Erlenmeyer flask with 0 mM and 5 mM sodium nitrate. For data below, normalized fluorescence intensity according to an optical density at 600 nm (OD600) are reported.

Figure 1. Expression of EGFP in non-induced (no nitrate) and induced (with 5 mM nitrate) cultures..

Figure 2. The fitted curve of EGFP expression in response to nitrate for PyeaR promoter.

Comparison of EGFP expression on different backbones

By cloning our nitrate sensor into different backbones (pSB1C3, pET28, and pUCIDT) and testing EGFP expression in response to different nitrate concentrations.

Figure 3. Fluorescence in response to different nitrate concentration for nitrate sensor in different backbones.

Conclusion

From our result, we can conclude that this nitrate sensor part can be used to detect nitrate at approximately 100 M nitrate and that this part is best used with a high copy number plasmid such as pUCIDT.