Team:Tsinghua/Engineering
Engineering Success
The diatomic gas nitric oxide(NO), a significant signal molecular in both eukaryotes and prokaryotes, has been demonstrated to regulate the formation of the biofilm in P.aeruginosa. The presence of small amount of indigenous NO could induce the dispersion of the biofilm as a response to anaerobic environment, with the activation of denitrification pathway. Moreover, it was demonstrated that P.aeruginosa could sense exogenous NO, leading to the biofilm dispersion as well. NO response pathway in Pseudomonas aeruginosa.has been revealed partially. NbdA protein on the cell membrane acts as an NO receptor and also has the phosphodiesterase(PDE) enzyme activity. Once it combines NO, PDE will be activated and leading to the degradation of bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP).Via uncertain pathways, the decreased c-di-GMP level triggers downstream gene expressions which may related to the improvement of the cell mobility and alternation of extracellular matrix, leading to the dispersion of biofilm.
Figure 1. Li Y, Heine S, Entian M, et al, 2013, Journal of bacteriology (2013)
Bacteria nitric oxide synthase (bNOS) from B.subtilis genome was applied as the source of NO. It could utilize L-Asp and oxygen as substrates, receiving the electron from NADPH and producing citrulline and NO. Compared with eukaryote NOS, bNOS lacks reductase domain therefore it requires additional reduction partner to function. It is assumed that engineered bacteria could be potential carriers of NO. Therefore, we wanted to clone the bNOS from B.subtilis and tried to expression in E.Coli.
We first cloned NOS gene in Bacillus subtilis by PCR (shown as Fig.1) and attached it to Pet28-a for determination using double restriction enzyme and DNA ligase. Then the ligated vector was transformed into the BL21(DE3) for protein expression and DH5α for plasmid application. The plasmid was sequencing by company, which was accord with our design.
Figure2. The result of NOS PCR from B.subtilis genome
Then we do the Western blot to make sure our system could express protein. The plasmid PET28-a itself contains a his-tag sequence, which is attached to the final of NOS sequence. So we can use antibody which can detect his-tag to detect the protein. To induce the production of target protein, the overnight culture at 37℃ was prepared. Arginine was added to 40ml LB(containing 100μg/ml kanamycin) to final 0.5mM concentration. ALA was added to final 0.45mM concentration and 400μl microbial was added. Then Incubate at 37℃ until OD600=0.5. Absorb half of the liquid and add it to the new conical flask as control. IPTG was added to the induced group to final 0.5mM concentration. All the groups were incubate at 28℃.The expression of 0-4h was detected. At the same time, GFP-His was added as the positive control.
Figure3. The result of western blot. The expression level of NOS-HIS gradually increased with the change of time.
In the next step we separately measured the change of NO over time in the NOS transformed BL21 (DE3) culture. The change of nitrite in the bacterial solution without IPTG induction with time was measured with total NO detection kit (Beyotime). We found that the nitrite content in the bacterial solution also decreased at the initial stage. This may be caused by the consumption of nitrite by the growth of bacteria, so we want to reflect the production of NO by the difference value of nitrite content between IPTG induced（IPTG+）and no IPTG induced（IPTG-）. The results are as followed.
Figure4. The difference in NO production by time.
As the figure illustrated below, the expression of NOS-HIS gradually increased with the change of time, indicating that our expression system works, which also explains why the production of NO gradually increased. Because in the first hours induced by IPTG, the protein expression was low, and the protein amount did not increase significantly until four hours.
For the last step, we tested our engineered bacteria in P.aeruginosa. Via the observation of laser confocal microscopy, the engineered bacteria successfully induced the dispersion of P.aeruginosa biofilm. (see more details in prove of concept)
Due to the pandemic and other accidents, our project is still needed to be perfected. However, the engineering success had brought us much confidence to present more wonderful results in the future.
Reference:
[1]Li Y, Heine S, Entian M, et al. NO-induced biofilm dispersion in Pseudomonas aeruginosa is mediated by an MHYT domain-coupled phosphodiesterase[J]. Journal of bacteriology, 2013, 195(16): 3531-3542.
[2]Pulin Liu Q H W C . Liu P, Huang Q, Chen W. Heterologous expression of bacterial nitric oxide synthase gene: a potential biological method to control biofilm development in the environment[J]. Canadian journal of microbiology, 2012, 58(3): 336-344.[J]. Canadian Journal of Microbiology, 2012, 58(3):336.
[3]Gusarov I, Starodubtseva M, Wang Z Q, et al. Bacterial nitric-oxide synthases operate without a dedicated redox partner[J]. Journal of Biological Chemistry, 2008, 283(19): 13140-13147.
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