Part:BBa_K5403012

Context iGEM Eindhoven 2024 created a library of parts (BBa_ K5403012 - BBa_K5403020) that encode fusion proteins that are designed to functionalize the membrane of the bacterial membrane vesicles (BMVs) of M. smegmatis and E. coli with a protein. These fusion proteins all consist of an N-terminal domain that is known to exist in the membrane of the BMVs, a flexible linker (BBa_K5403002) and a C-terminal GFP (BBa_K5403997). GFP was chosen for initial characterization, but the part contains an NheI-restriction site that allows you to replace the GFP with a gene for any protein of your interest.

Part description This fusion protein is based on the outer membrane protein A (OmpA) that is present in the membrane of E. coli. OmpA has been used as an anchor to attach other proteins to the membrane by recombinant expression of a fusion protein (Beck et al., 2017). In this fusion protein, only the 21-amino-acid signal sequence at the N-terminus (described in Movva et al., 1980) was used, with the hypothesis that only this signal sequence will be enough to anchor a protein in the membrane.

The protein was expressed in BL21 cells and analyzed by FACS with a primary and a secondary antibody. The results are shown below.

The OmpA(N21) membrane protein, fused to GFP, is expressed in BL21 cells by induction of IPTG. The sample is stained with a secondary antibody conjugated to a dye, to increase the fluorescent signal of the GFP. The fluorescent signal is analysed with FACS.

Aim: Analyze the presence of membrane protein OmpA(N21), bound to GFP, on BL21 cells.

Methods: To analyze the expression of both membrane proteins, a large culture of 2YT medium with BL21 cells transformed with a PET28a plasmid was made. The culture was grown until a OD600 of ~0.6 was reached. At that point, IPTG was added to the culture to induce protein expression and the large culture was incubated at a lower temperature of 18 ° overnight. The protocol can be found here

In the first setup of this experiment, the IPTG incubated large culture was directly analyzed with FACS. These FACS results showed little to no GFP signal. The experiment was repeated, but still no signal was visible. To overcome this issue, an antibody staining was introduced to increase the signal.

Antibody staining was done with a primary antibody for GFP and a secondary antibody conjugated to a Cyanine3 dye. Several negative controls were incorporated into the experiment:

- Negative control without IPTG induction. Used to conclude that the protein is not expressed when IPTG is not present. - Negative control without antibody staining. Used to conclude that antibody staining is necessary for visualization of the GFP signal. - Negative control without IPTG induction and without antibody staining. Used to determine a threshold for the signal noise.

-Negative control with only secondary antibody incubation. Used to analyze the occurence of non-specific staining.

Figure 1: FACS result - Sample OmpA(N21) gated. Figure 2: FACS result - Sample OmpA(N21) Single cells. Figure 3: FACS result - Sample OmpA(N21) signal.

To analyze FACS results, the data has to be gated by hand. By gating, unwanted data that would interfere with the analysis is exluded. This reduces noise and improves data accuracy. Gating is done based on the forward and side scatter of the sample. In Figure 1, the gating is shown.

To improve accuracy even more, doublet discrimination is used. Doublets are events where two cells pass through the flow cytometer's laser at the same time or very closely together, producing an artifact in the data. These doublets can be mistakenly counted as a single event, leading to inaccurate results. This discrimination is done by comparing pulse height (FSC-H) and pulse area (FSC-A) in Figure 2.

When both gating and doublet discrimination have been completed, the quartiles lines that seperate the quartiles can be determined. It is adviced that, in a negative control, maximum 1% of our signal falls in Q1 and Q2. Figure 29 shows the correct seperation.

Now that the FACS data is completely filtered, the settings for the gate, doublet discrimination and placement of the quartile lines can be repeated on all samples for OmpA.

Figure 4: FACS result of OmpA(N21) (no IPTG and no antibody staining). Figure 5: FACS result of OmpA(N21) (with IPTG, but no antibody staining). Figure 6: FACS result of OmpA(N21) (with IPTG and only secondary antibody incubation).

Figure 30 shows the signal of the OmpA(N21) sample without IPTG and with no antibody staining. It is expected that the signal will not be visible in Q1 and Q2, which is confirmed by the FACS result. Figure 31 shows the signal of the OmpA(N21) sample with IPTG, but without antibody staining. The signal will therefore only come from the protein-bound GFP. As can be seen in the Figure, around 99% of the signal is visible in Q4 and Q3. This suggests that the GFP itself is not sufficient enough for FACS analysis and the antibody staining is necessary. Figure 32 is the signal of the OmpA(N21) sample with IPTG and only secondary antibody. As only around 1.5% of the signal is present in Q1 and Q2, it can be concluded that no staining occured. Therefore, it can be concluded that little to no non-specific secondary antibody staining occured.

Figure 7: FACS result of OmpA(N21) (with IPTG and antibody staining). Figure 8: FACS result of OmpA(N21) (without IPTG, but with antibody staining).

For the OmpA(N21) sample with IPTG and antibody staining, around 9% of the signal is within the color spectrum of the dye (Q1) in Figure 33. This is larger than the sample without antibody staining.

When looking at Figure 34, around 4% of the signal is present within the color spectrum of the dye. This is not expected, as no IPTG is added to this sample, meaning that the protein of interest should not be expressed. As the negative control has proven that non-specific staining does not occur, another explanation must be found. One could be leaky expression.

Leaky expression in protein expression refers to the unintended, low-level production of a protein even when the gene or promoter that controls its expression is supposed to be repressed or inactive [4]. In other words, the regulatory system designed to tightly control protein expression is not fully effective, allowing for some leakage of expression in the absence of the intended inducer or under non-inducing conditions. The fact that the signal is present at a lower quantity than in the IPTG induced sampe confirms this hypothesis.

Conclusion: Although some leaky expression occurs in the non-induced sample, a clear staining signal can be seen in the induced OmpA(N21) sample. As non-specific staining is ruled out, it can be concluded that the OmpA(N21) bound to GFP is correctly expressed in the membrane of BL21 cells.

References Beck, B. R., Lee, S. H., Kim, D., Park, J. H., Lee, H. K., Kwon, S., Lee, K. H., Lee, J. I., & Song, S. K. (2017). A Lactococcus lactis BFE920 feed vaccine expressing a fusion protein composed of the OmpA and FlgD antigens from Edwardsiella tarda was significantly better at protecting olive flounder (Paralichthys olivaceus) from edwardsiellosis than single antigen vaccines. Fish & Shellfish Immunology, 68, 19–28. https://doi.org/10.1016/j.fsi.2017.07.004 Movva, N.R. et al. (1980) Amino acid sequence of the signal peptide of ompA protein, a major outer membrane protein of Escherichia coli. (1980, 10 januari). PubMed. https://pubmed.ncbi.nlm.nih.gov/6985608/