Part:BBa_K2967030

NorR-PnorV-amplicp

When nitric oxide is present in the environment, the promoter PnorV will initiate the expression of the blue chromoprotein.

Last year, the NO sensor had a serious 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. 1B), 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. 1A). After adding the terminator, we found the amilCP leakage problem has been significantly relieved (Fig. 1C).

Figure 1A. 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.

Figure 1B. 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.

Figure 1C. 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.

References

[1] Rachmilewitz D, Stamler J S, Bachwich D, et al. Enhanced colonic nitric oxide generation and nitric oxide synthase activity in ulcerative colitis and Crohn's disease[J]. Gut, 1995, 36(5): 718-723.

[2] Ljung T, Herulf M, Beijer E, et al. Rectal nitric oxide assessment in children with Crohn disease and ulcerative colitis. Indicator of ileocaecal and colorectal affection[J]. Scandinavian journal of gastroenterology, 2001, 36(10): 1073-1076.

[3] Tucker, N. P., D’Autreaux, B., Yousafzai, F. K., Fairhurst, S. A., Spiro, S., and Dixon, R. (2008) Analysis of the nitric oxide-sensing non-heme iron center in the NorR regulatory protein. J. Biol. Chem. 283, 908−918.

[4] Bush, M., Ghosh, T., Tucker, N., Zhang, X., and Dixon, R. (2011) Transcriptional regulation by the dedicated nitric oxide sensor, NorR: a route towards NO detoxification. Biochem. Soc. Trans. 39, 289−293.

[5] Archer, E.J., Robinson, A.B. & Suel, G.M. Engineered E. coli that detect and respond to gut inflammation through nitric oxide sensing. ACS Synth. Biol. 1, 451–457 (2012).

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 AgeI site found at 867
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 642