In recent years, targeted drugs represented by monoclonal antibodies have greatly improved the clinical treatment effect of inflammatory bowel disease, but there are unmet needs such as high prices, unresponsiveness of some patients, infection caused by long-term medication, and poor patient compliance. In this project, EcN will be genetically engineered: the nucleotide sequences of anti-TNFα nanobodies (A) will be synthesized, with both ends of Ozoralizumab(A) and nanobody-Ab V565 (B) modified to include a 6×His Tag for subsequent purification and characterization. The N-terminus of A is appended with a signaling peptide to enhance the secretion of the nanobody into the extracellular space(Figure 1). The synthesized sequences are then cloned into a transformation vector and introduced into EcN1917 through transformation. Clindamycin-resistant colonies are selected to ensure stable integration of the edited EcN1917 strain. Select multiple clones for further characterization.

Figure 1: Summary of the plasmid construction and engineered EcN 1917 bacterial

The subsequent characterization encompasses the following steps:

Cycle 1: p15A-Ozoralizumab Design and Build

Cycle 2: p15A-V565 Design and Build

Cycle 3: Test

Test1: Enzyme-linked immunosorbent assay(ELISA1) and Western Blot (WB)

Test2: ELISA2 and cell function test,

Test3: Improved verification from molecular level to cell level

Cycle 4: learn

Design：

Initially, we chose to perform the in-frame assembly of the plasmid in DH5α cells, followed by the transformation of the successfully assembled plasmid into EcN1917 cells. The gene sequences for both the A and B antibodies were synthesized using gene synthesis techniques. To start, we constructed the nanoAb-A plasmid through homologous recombination.(Figure 2).

Figure 2: Snapgene Plasmid and sequence diagram of nano-Ab A

Build:

We obtained the bacterial colony through solid plate culture overnight and we subsequently dispatched them to a biotechnology firm for sequencing (Figure 3 A ). The results in the following two images were obtained (Figure 3 B ). Since they are solid lines, there were no base mismatch for gene mutations, indicating the success of the construction work.

Figure 3: Colony culture of DH5α-p15A-A and Sanger sequencing result.

This image depicts a culture dish in which the target genes A were successfully transformed into EcN 1917. The bacteria labeled with -1 on the left are diluted by a factor of 100, whereas those on the right remain undiluted. Figure 4 illustrates that the colony count of p15A-A bacteria significantly decreased upon 100-fold dilution, while an increased number of colonies was observed in the undiluted sample.

On the right side is the validation of monoclonal colonies. According to the image, the length is between 800-1200bp, with a specific length of 1088bp. The length is consistent with the target gene, indicating successful transformation.

Figure 4: Colony culture of transformed EcN1917 and colony PCR verification

Design：

Building on the findings from cycle 1, we utilized the synthesized DNA sequence of the B antibody to develop and validate the nanoAb-B plasmid. This involved constructing the plasmid and confirming its functionality through a series of validation steps.(Figure 5).

Figure 5: Snapgene diagram of p15A-nanoAb-B plasmid

Build:

We obtained the bacterial colony through solid plate culture overnight and we subsequently dispatched them to a biotechnology firm for sequencing (Figure 6A ). The results in the following two images were obtained (Figure 6B ). Since they are solid lines, there were no base mismatch for gene mutations, indicating the success of the construction work.

Figure 6: Colony culture of DH5α-p15A-B and Sanger sequencing result.

Figure 7 illustrates that the colony count of p15A-B bacteria significantly decreased upon 100-fold dilution, while an increased number of colonies was observed in the undiluted sample. In contrast, for the p15A-B gene subjected to 100-fold dilution, there was virtually no observable growth of colonies. On the right side is the validation of monoclonal colonies. According to the image, the length is between 800-1200bp, with a specific length of 1088bp. The length is consistent with the target gene, indicating successful transformation.

Figure 7: Transformation of the p15A-nanoAb-B plasmid to EcN1917 and colony PCR verification

Test 1: expression verification by WB and ELISA (TNFα-coated plate)

We successfully constructed the EcN1917-nanoAb-A and EcN1917-nanoAb-B strains, which were cultured at 16°C and 37°C, respectively, in LB and SB media for a duration of 48 hours. Following centrifugation and subsequent filtration through a 0.22 μm membrane to eliminate bacterial cells, we obtained the final antibody product(Figure 8). The expression of the nanoAb-His fusion protein was effectively detected via Western blotting. We coated the assay plate with TNFα and employed an Anti-His-HRP ELISA to detect the antibody, yielding a significant light signal. By integrating these two experimental approaches, we preliminarily conclude that the supernatant from the engineered probiotic culture contains nanobody proteins, with nanobody B exhibiting higher expression levels than nanobody A.

Figure 8: Expression verification of nanoAb-A and nanoAb-B by Western blot and ELISA (coated with TNFα)

Note: The PBS is the blank control group and the Posi-Ab means positive control group. AL means desired gene A in LB medium, AS means gene A in SB medium at 37 °C, BL means gene B in LB medium at 37 °C, BS means gene B in SB medium at 37 °C, AL16 means gene A in LB medium A at 16 °C, AS16 means gene A in SB medium at 16 °C, BL16 means gene B in LB medium at 16 °C, BS16 means gene B in SB medium at 16 °C.

Test 2: ELISA2 to investigate the blocking effect from molecular level of TNFα and TNFR

Using the nanobody supernatant collected in Test 2, we coated TNFR onto the plate and measured the quantity and affinity of TNFα bound to it using an Anti-TNFα-HRP detection antibody. The positive control group consisted of a commercially available anti-TNFα antibody, while the experimental group included eight different nanobody supernatants. PBS served as the negative control. We then evaluated the blocking efficacy at varying TNFα concentrations. Notably, the AL, AL16, and AS16 groups demonstrated significant blocking capabilities at both 0.5 ng/mL and 1 ng/mL TNFα concentrations(Figure 9).

Figure 9: ELISA (coated wit TNFR) verify the blocking effect of nanoAb on the binding of TNFα to TNFR.

Note:The PBS is the blank control group and the Posi-Ab means positive control group. AL means desired gene A in LB medium, AS means gene A in SB medium at 37 °C, BL means gene B in LB medium at 37 °C, BS means gene B in SB medium at 37 °C, AL16 means gene A in LB medium A at 16 °C, AS16 means gene A in SB medium at 16 °C, BL16 means gene B in LB medium at 16 °C, BS16 means gene B in SB medium at 16 °C.

Test 3: Cell viability test to verify the protect effect of nanobody antibody on cell

After TNF-a binds to the TNF receptor (TNFR) on the cell surface, it initiates apoptosis and necrosis programs, ultimately leading to cell death. However, after TNFα binds to the nanoantibody, it can no longer bind to TNFR, and therefore cannot cause cell death. Therefore, we further studied the effect of nanoantibodies in inhibiting inflammatory factors through cell experiments.

TNF-α was mixed with a series of nanoantibodies at different concentrations (a total of 5 nanoantibodies were obtained by culture, and the concentration of each nanoantibody was reduced by half in turn, and the mixture of TNF-α and PBS was used as a negative control) and incubated with U937 cells for 6 hours. The viability of each group of cells was then detected using the CellTiter-Lumi kit. The results (Figure 10) showed that compared with the control group, the nanoantibodies obtained by the 5 culture methods all showed a good effect in inhibiting cell death. And this effect is positively correlated with the concentration of the nanoantibody, that is, as the concentration increases, the effect of inhibiting cell death increases.

Figure 10: Cell viability test by Cell-titer assay to verify the protect effect of nanoAb on cell stimulated by TNFα.

1. The expression level of Ozoralizumab (A ) can be further improved during the experiment, and WB experiment is a little fuzzy, we can run a better WB diagram. At the same time, ELISA experiments can be done more gradient protein concentration verification.

2. In the future, the iGEM team can further explore and optimize p15-B, for example, the B-carrier protein expression is not good, which requires more experiments to optimize it.

3. Other iGEM teams can also further optimize the dual-target nanobodybased on our project.They can use nanobodies in other fields.