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From 2014.igem.org

Proof of Principle

Microscopy

Using three constructs (BBa_I20260,BBa_K523013, and our biobrick BBa_K1423005) we used fluorescence microscopy to test the efficiency of cell surface localization of fluorescent proteins using two cell surface anchoring motifs :BclA and INP. We hypothesized that BclA would be a more efficient cell surface protein anchoring motif than INP. The E. coli containing each of the constructs were incubated overnight at 37degreesC with shaking. 20µL samples of each culture were dropped onto microscope slides and allowed to air dry. Slides were then fixed in methanol for 10 minutes, dried, and wet mounted with a coverslip in a 50% glycerol solution. Slides were imaged on a Zeiss AxioVert miscoscope with an 100x oil immersion lens, and imaged with a Zeiss AxioCam MRm camera. Images were acquired using Zen software, and processed with Adobe Photoshop. All images were exposed for 2 seconds. Brightness and contrast adjustments made during processing were applied equally to all panels within the figure.

We first analyzed an internal expression of GFP using BBa_I20260. Figure 1 below shows the images captured from the microscopy. The figure shows that the YFP is being expressed throughout the entire cell indicating that it is not localized to the cell surface. The image on the left is E. coli expressing GFP internally at 100X magnification and the image on the right is this same image contrast normalized and zoomed in on a few E. coli.

Figure 1: Internal Expression of GFP: Original Image 100X (left) and Contrast Normalized and Zoomed (right)

We then analyzed YFP cell surface expression using an Ice Nucleation Protein-YFP biobrick created by Edinburgh team in 2011 (BBa_K523013). The microscopy images shown below in Figure 2 show sporadic expression of YFP around the outside of the cell, with sparse fluorescent dots in the middle and heavier expression on the dipoles.You can see the E. coli expressing INP linked to YFP also have an abnormal morphology in that they are elongated, most likely due to a fission defect.The image on the left is E. coli expressing YFP externally using INP for cell surface localization at 100X magnification and the image on the right is this same image contrast normalized and zoomed in on the E. coli.

Figure 2: External Expression of YFP Using INP: Original Image 100X (left) and Contrast Normalized and Zoomed (right)

Lastly, we analyzed YFP cell surface expression using our BclA-YFP biobrick (BBa_K1423005) to localize expression to the cell membrane. The microscopy seen below in Figure 3 exhibits that the expression of YFP using BclA is nearly continuous around the outside of the cell and that the cell has a normal E. colimorphology. This supports our hypothesis that BclA is a more efficient cell surface anchoring motif than INP. The image on the left is E. coli expressing YFP externally using BclA at 100X magnification and the image on the right is contrast normalized and zoomed in on the E. coli.

Figure 3: External Expression of YFP Using BclA: Original Image 100X (left) and Contrast Normalized and Zoomed (right)

It is important to address that in all three figures above, there is a heavier expression of YFP at the dipoles. This is called dipole protein aggregation and occurs in E. coli when a single protein is over-expressed. This dipole expression of YFP is expected, so it will not have an impact on our agglutination assay because the protein is still successfully extracellularly expressed.

Western Blot

We attempted another proof of principle experiment, in order to support the fact that BCLA causes cellular surface expression of proteins. To do this, we performed a fractionation experiment followed by a Western Blot. We were doing the fractionation to isolate the membrane bound proteins from the cytoplasmic proteins. We then performed a Western Blot using a GFP antibody. Our results suggest that their might be some localization of YFP to the cell surface membrane using the BclA-YFP construct. However, the results were not conclusive. We believe that the Figure 4 below shows the localization of the YFP to the membrane. This is the case because in the BclA-YFP well in the membrane bound protein lane there was an extremely high expression on the blot. This shows that the YFP is being localized to the cell surface on the biochemical level. When performing the Western Blot, it appeared that the wells were loaded evenly but a loading control was not used which is part of the reason we concede these results to be mildly inconclusive. We still feel the results of this Western Blot adds extra support to the efficiency of BclA cell surface protein localization when combined with our microscopy results.

Figure 4: Cell Fractionation and Western Blot Results

Agglutination Assay

For our proof of principle agglutination assay, we wanted to test if antigens expressed on the cell surface, in this case YFP antigens, would bind to their corresponding antibody and produce visible agglutination. In order to do this, we combined in a well plate GFP antibodies (which is analogous to YFP antibodies) with E.coli expressing YFP externally through the use of both INP and BclA. As a control, we also mixed GFP antibodies with E.coli internally expressed GFP as well as E.coli externally expressing the CAEV antigen using our biobrick(BBa_K1423004).

If our agglutination experiment is successful, we would expect agglutination in the wells with E.coli externally expressing YFP (using BCLA and INP). We would not expect agglutination in the well with E.coli internally expressing GFP because the GFP is internal, meaning the extracellular antigen cannot bind to it. We would also expect no agglutination in the well containing E.coli externally expressing the CAEV antigen because that is the incorrect antigen, and it is not recognized by the GFP antibodies.

It is important to first establish how to interpret the results of our agglutination assay. When agglutination occurs, antibodies will bind to their matching antigen pairs, creating a mat on the bottom of the well. This is the case because each antibody has two antigen binding sites, meaning it can bind to more than one antigen and form an interwoven antigen-antibody complex. No agglutination, on the other hand, would manifest as a small dot at the bottom of the well, because no interwoven antigen-antibody complexes were formed and the cells just clumped to the bottom due to gravity. A schematic showing the differences between a positive agglutination and a negative agglutination result can be seen below.

Figure 5: Agglutination Schematic- An unmatching antibody on top and a matching antibody below that; with the final results on the bottom. The final results are a top view of the containing wells.

The actual results of our assay can be seen below in Figure 6. It can be seen that the wells with cellular surface expression of GFP (the bottom two rows) were positive (a mat) as expected while the controls (the top two rows) were not agglutinated and had a dot of cells in the bottom of the well. This proves that it is possible to see a visual confirmation of agglutination.

Figure 6: Agglutination Assay Results