"

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 GFP 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 GFP 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.

Agglutination Assay

We were able to successfully achieve the results we wanted with the Agglutination Assay. When antibodies are in the presence of their matching antigen pairs, they will bind to each other. Antibodies have two antigen binding sites which allows a network of antibody-antigen expressing bacteria to form. This network manifests on a large scale as a mat of bacteria on the bottom of the containing vessel, as opposed to a solid dot formed by unagglutinated antibodies. We based our assay off of this paper.

For our proof of principle agglutination, we used surface-bound YFP (Yellow Fluorescent Protein) and a corresponding GFP (Green Fluorescent Protein)/YFP antibody (the part of the protein that the antibody binds to is not affected by the part that causes the color change.) We used both of the surface expression proteins we had at our disposal: BclA (the one we made) and INP (a pre-existing biobrick). As controls, we used internally expressed YFP as well as a protein that was externally expressed but did not match with the antibody (we used the CAEV protein we made).

This photo clearly shows that the wells containing the bacteria with the matching antigen on the surface formed agglutinated mats while the control wells did not agglutinate and formed dots at the bottom of the wells.

Western Blot

We attempted a third proof of principle experiment, but the results were not optimal. 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 to the cell surface membrane of the YFP within the BclA-YFP construct; however, the results were not conclusive. We believe that the image below shows the localization of the YFP to the membrane. 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 when combined with our microscopy and agglutination results.

The BclA-YFP section under the membrane bound protein lane shows extremely high expression on the blot. This shows that the YFP is being localized to the cell surface on the biochemical level.