Part:BBa_K2967026
The yeaR promoter added an extra NsrRBSs-RBS-Luc
123
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1478
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