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Nitric Oxide Sensing Genetic Circuit

This composite part contains the nitric oxide sensor pNorVß (BBa_K4387000), followed by 2 different ribosome binding sites. The promoter induces upon NO binding the expression of a monovalent anti-TNFα nanobodoy BBa_K4387996. The additional NorR at the end of the composite part enhances the positive feedback-loop, increasing the response of the promoter to NO. [1] Together with the hemolysin A secretion machinery BBa_K4387987, a complete genetic circuit is obtained that allows secretion of nanobodies [2] or if exchanged, secretion of other proteins of interest.

Sequence and Features

Characterization

Western blot

Figure 1: Double transformed E. coli Nissle 1917, able to secrete the monovalent nanobody VHH#2B, were induced with different DETA/NO concentrations (0mM, 1mM and 2mM) and incubated at 37°C overnight. Anti-myc antibodies were used in this Western blot to stain secreted nanobodies in the bacterial supernatant.

We double transformed our chassis, the probiotic E. coli Nissle 1917, with the high copy plasmid containing this composite part required for induced nanobody expression, and the medium copy number plasmid containing the composite part BBa_K4387987 needed for the secretion system. Liquid overnight cultures of transformed bacteria were grown and induced by adding 2mM, 1mM or 0mM NO respectively to the cultures. DETA/NO was used as a nitric oxide source for the induction experiments. On the next day, the cells were centrifuged, and the supernatant was run on a gel. To see if nanobodies of the correct size have been secreted by the bacteria, we conduct a Western blot by detecting the myc-tag fused to the nanobodies (Figure 1).

As seen in figure 1, we received a band with the size of approximately 45 kDa which fits the expected size of the monovalent nanobody candidate VHH#2B together with the myc-tag and HlyA-tag. We can therefore assume that the bacteria were able to secrete whole nanobodies.

However, the first two bands showing the bacterial samples that have not been induced with DETA/NO and therefore should not have secreted nanobodies are visible, indicating that the promoter is leaky. To investigate further, we compared the intensity of the bands that we received from the Western blot with imageJ. For each condition a numerical average was calculated:

image J values

On average the bands from the 2mM DETA/NO are 41% more intense than the control indicating an increased protein secretion upon DETA/NO induction. However, the non-induced expression appears to be quite leaky. A possible explanation for the leakiness might be the two ribosomal binding sites that follow the promoter, leading to an enhance promoter activity but also to more leakiness. Additionally, the bacterial cultures were grown overnight for about 15 hours at 37°C, leading to a dense E. coli culture. It is possible that over time nitric oxide might have been metabolically produced by the bacteria and accumulated, leading to an increasing self-induction over this long period of time.

Endpoint Flow Cytometry Assay

We additionally analysed our induced bacteria via flow cytometry: Cell cultures were grown overnight in LB medium supplemented with antibiotic, diluted in 2mL of M9 (supplemented with glucose, cas amino acids and an antibiotic) in a 1:10 ratio (v/v), induced with different NO concentrations and grown for 7 hours in a shaker (37°C, 220 RPM). Samples were then chilled in ice to halt cell growth and diluted in 1mL of cold PBS (1:500 v/v ratio). A total of 100,000 cells per sample was measured in a BD FACSCanto II flow cytometer (FSC: 625V, SSC: 420V, FITC: 650V, Event threshold: FSC & SSC > 200, Channel: FITC (λEx 488 nm / λEm. 530/30 nm, High flow rate: ~ 10,000 events/s).

Figure 2a: Violin Plots and Boxplots for Our 6 Different Constructs. These plots represent the distribution of FITC-H (green fluorescence) values for our six different plasmids at four different concentrations of DETA/NO. Each measurement consisted of 100'000 cells.

Figure 2b: Noise Levels of Our 6 Different Constructs. Noise levels are measured as the coefficient of variation for construct sample and NO concentration. Most constructs show low noise levels at high concentrations.

While figure 2a shows that the construct with pNorVβ and 2 RBS (BBa_K4387006) has higher overall GFP expression values, it is also leakier than this construct with pNorVβ and 1 RBS (BBa_K4387005) or the construct with pNorVβ and 3 RBS (BBa_K4387007). If high GFP expression is required, but some leakiness does not matter much, we recommend choosing BBa_K4387006. If lower leakiness is essential, but GFP expression does not need to be very high, we recommend using parts BBa_K4387005 or BBa_K4387007 instead.

ELISA

Figure 3: ELISA testing TNFα-binding capabilities of the secreted monovalent nanobody VHH#2B obtained from E. coli Nissle 1917 after nitric oxide induction, compared to purified and secreted nanobodies form MC1061

To prove that the secreted nanobodies not only have the correct size but are also able to elicit their TNFα-binding abilities, we performed an ELISA (Figure 2). Adalimumab, a monoclonal anti-TNFα antibody already used in the clinics to treat IBD patients, served as a positive control (wells C1-2), and a sybody against a membrane protein was the negative control (wells C3-4). We could show that the transformed E. coli Nissle 1917 is able to secrete functional anti-TNFα nanobodies upon nitric oxide induction (row A).

References

• [1] Xiaoyu J. Chen et al., 2021, Rational Design and Characterization of Nitric Oxide Biosensors in E. coli Nissle 1917 and Mini SimCells
• [2] Silence, Karen, Lauwereys, Marc, De Haard, Hans, et al. "Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor", Int. Publication Number: WO 2004/041862 A2, 21 May 2004