Revision as of 05:38, 5 October 2019

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

This year, we chose BBa_KK2967017 (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 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.

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.

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.

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.

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.

Sequence and Features

Sequence and Features