Difference between revisions of "Part:BBa K2817000"

Latest revision as of 11:29, 10 October 2019

PnorV-RBS-amilCP

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

Usage and Biology

Engineered bacteria could be an ideal way to treat IBD. First, we need an appropriate sensor to sense the inflammatory signal and initiate the release of the drug. After comparing the inflammatory sensors used in some iGEM teams, we selected the inflammatory sensor based on nitric oxide molecules used by ShanghaiTechChina_B 2016 team. Nitric oxide is a natural signaling molecule of inflammation, and the concentration of intestinal inflammation in patients with IBD is significantly increased. It has been reported that the average concentration of nitric oxide in the rectum of normal people reaches 60 nM, while in patients with IBD it reaches 5.5 μM, nearly 100 times higher than that of the former. Therefore, the nitric oxide molecule is an ideal input signal for engineered bacteria, allowing the engineered bacteria to function only at the target location, thereby avoiding inappropriate output.

There are some natural nitric oxide sensors in E. coli, in which the enhancer binding protein NorR is specific and can only react with nitric oxide and cannot interact with other nitrogen forms. NorR binds to three conserved sites on the NorV promoter (PnorV) via the C terminal HTH domain. When nitric oxide is absent, the N terminal domain GAF of NorR blocks the central domain AAA+ to inhibit its binding to the transcription factor σ 54, and thereby prevent transcription of the nitric oxide reductase NorV. When nitric oxide binds to the GAF domain of NorR, it will release the AAA+ domain, allowing σ 54 to initiate transcription of NorV.

In our inflammation sensor, we used two parts which are NorR and PnorV. amilCP and GFP were selected as our reporter. Although the E. coli host has native NorR expression, increasing its expression facilitates the elimination of stoichiometric imbalances between NorR in the genome and PnorV on the foreign plasmid, avoiding interference with the host's release of nitric oxide. We used the double expression plasmid pCDFDuet-1 to construct our sensor module. Placing NorR under the control of the first T7 promoter, we are able to regulate its expression by IPTG induction. We also replaced the second promoter with PnorV and added the reporter gene downstream which allows us to activate the expression of the reporter by adding nitric oxide.

We transformed the constructed plasmid with NO sensor into DH5α, cultured at 37 ℃ overnight, and then diluted to OD600 = 0.2. Culturing bacteria at 37 ℃ for 1.5 hours, the appropriate concentration of inducer IPTG and SNP aqueous solution were added. After 6 hours of culturing, 1 mL of the bacterial solution was centrifuged at 8000 r.p.m for 1 min (Figure 1). It can be seen that the NO released by the SNP aqueous solution can effectively activate the expression of the reporter gene. Surprisingly, the blue chromoprotein in group 1mM IPTG without SNP was also be activated with undetermined mechanisms. We speculated that it may be caused by leakage expression of promoter PnorV when the NorR is overexpressed. Due to this reason, we would use a weak promoter to express NorR in further engineered bacteria.

http://219.216.82.193/cache/7/04/2018.igem.org/cf58bf3e68fe4e92fa0889b84a5b3539/T--NEU_China_A--results-2.png

Figure 1. Pellets of bacteria transformed with constructed NO sensor plasmid after induction of 6h. 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.

[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).

NEU_CHINA 2019

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.

More sequence detail about our improvement please see our new part, BBa_K2967030.

Sequence and Features