Part:BBa_K5403013

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.

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).

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 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, bound to GFP, on BL21 cells.

Methods: The sample of OmpA bound to GFP is prepared and analyzed in the same way as the OmpA(N21) sample: antibody staining and analysis with FACS. The protocol can be found here

Results:

The preproccesing of the FACS results is done in the same way as for the OmpA(N21) sample. First, the results are gated correctly. After this, doublet discrimination is introduced. Lastly, the placement of the quartile lines are determined.

Figure 1: FACS result of OmpA (no IPTG and no antibody staining). Figure 2: FACS result of OmpA (with IPTG and no antibody staining). Figure 3: FACS result of OmpA (with IPTG and only secondary antibody incubation).

Figure 2 shows that, with IPTG induction, the GFP signal itself is not strong enough for FACS analysis. This is a result that would also be expected for the sample without IPTG induction, but the pattern of this sample in Figure 1 is differen from the sample with IPTG. An explanation for this could be that IPTG induction results in misfolding or degradation of OmpA. The expression of the OmpA could also potentially lead to toxicity in the cell, preventing GFP expression from being detectable.

Figure 3 shows the negative control sample for non-specific binding of the secondary antibody. When non-specific staining does not occur, no signal is expected in Q1. But it in the Figure it is visible that 14% of the total signal is in Q1, suggesting a Cyanine3 staining. This suggests that the secondary antibody not only binds to the primary antibody, but also somewhere else on the BL21 cell membrane.

Figure 4: FACS result of OmpA (with IPTG and antibody staining). Figure 5: FACS result of OmpA (without IPTG, but with antibody staining).

When looking at Figure 4, around 27% of the sample shows antibody staining. The intensity of this signal closely resembles that of the secondary antibody control group. It is therefore difficult to conclude if this is staining from the antibody complex binding to GFP or non-specific binding of the secondary antibody.

Figure 5, the sample without IPTG, shows a different signal pattern. Although the total amount of signal is lower than in Figure 4, the intensity is higher. The presence of signal could be due to leaky expression, but there is no clear explanation on why the signal intensity is much higher compared to the induced sample.

Conclusion: The antibody staining of OmpA + GFP shows non-specific secondary antibody binding. This non-specific staining makes it difficult to confirm the presence of OmpA + GFP on the membrane of the BL21 cells, especially because the signal of the induced sample shows a similar pattern as the secondary antibody negative control. On top of that, the non-induced sample shows a higher intensity than the induced sample, which cannot be explained by leaky expression alone.

Due to these uncertainties, it is adviced to redo the exact experiment to check if the result is the same. If this results in the same FACS data, a different combination of primary and secondary antibody can be chosen to hopefully reduce non-specific secondary antibody binding.

Next to the FACS experiment, it is adviced to perform other experimental set-ups to identify the presence of the OmpA membrane protein. These could be visualization experiments, like super-resolution microscopy (SRM) and immunofluorescence microscopy (IF), or another labelling method like cell surface Biotinylation. Next to that, the induction system itself can be tested by using qPCR for mRNA levels or Western blot for protein levels.

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