Team:NEU CHINA/improve.html

Improve

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.

PARTS

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 obtain low basal level 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.

Figure 4A. Diagram for NO sensor system in pCDFDuet-1 plasmid. T7 promoter, the gene downstream of this promoter will be transcribed when there is T7 RNA polymerase. lacO, the sequence represses the nearby promoter when there is no inducer (e.g. IPTG). RBS, ribosome binding site. NorR, NO binding protein. PnorV, a promoter which is sensitive to NO. amilCP, blue chromoprotein.

REFERENCES

[1]. Merulla, D. & van der Meer, J. R. Regulatable and modulable background expression control in prokaryotic synthetic circuits by auxiliary repressor

547–567 (2013).

[2]. Fernandez-Rodriguez, J. & Voigt, C. A. Post-translational control of gencircuits using Potyvirus proteases. Nucleic Acids Res. 44, 6493–6502 (2016)

PARTS IMPROMENTS

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]

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.

As for solving the serious NO sensor（NorR-pnorv-amplicp）leakage problem last year. At first , we considered the NorR over expression might be the key of the leakage. However, after we knock out the NorR, the leakage is more serious (Fig. 4B), and it seems that the NO sensor is out of work. So, we predicted that the plasmid constructed last year leaks a terminator downstream the NorR sequence. Therefore, we added terminator B0010/B0012 to the inflammation sensor we constructed (Fig. 4A). After adding the terminator, we found the amilCP leakage problem has been significantly relieved (Fig. 4C).

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.

REFERENCES

[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.

Figure 4B. Pellets of bacteria transformed with constructed NO sensor plasmid after 4hr induction at 37 ℃. From left to right: control, 0.5mM IPTG without SNP, 1mM IPTG without SNP, 100μM SNP,0.5mM IPTG with 100μM SNP, 1mM IPTG with 100μM SNP. From top to bottom: empty vector, T7-Norr-PnorV-amplicp,T7-PnorV-amplicp.

A

B

Figure 2. The response to NO sensors.

Figure 4C. Pellets of bacteria transformed with constructed NO sensor plasmid after 2hr induction at 37 ℃. From left to right: control, 0.5mM IPTG without SNP, 1mM IPTG without SNP, 0.5mM IPTG with 100μM SNP, 1mM IPTG with 100μM SNP. From top to bottom: empty vector, T7-Norr-PnorV-amplicp, T7-T-PnorV-amplicp.

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.

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.