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From 2014.igem.org

Proof of Principle

Microscopy

Using three of the constructs we created we tested the cell surface localization of YFP by BclA and INP using fluorescence microscopy. The E. coli containing each of the constructs were incubated overnight at 37C 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. The first construct analyzed was internal expression of YFP. The following are the images captured from the microscopy. The first construct analyzed was internal expression of YFP. The image shows that the YFP is being expressed throughout the entire cell indicating that it is not on the cell surface. The first image is E. coli expressing YFP internally at 100X magnification. The second image is this same image contrast normalized and zoomed in on the E. coli.

The next construct used an ice nucleation protein to anchor the YFP in the cell membrane. The microscopy image shows sporadic expression of YFP around the outside of the cell. The E. coli expressing INP linked YFP have an abnormal morphology in that they are elongated. The expression of YFP shows some sporadic dots around the middle and heavier expression on the dipoles. The first image is E. coli expressing YFP externally using INP at 100X magnification. The second image is this same image contrast normalized and zoomed in on the E. coli. The INP-YFP construct used for this construct was made by the Edinburgh iGEM team of 2011 and is named BBa_K523013 in the iGEM parts registry.

The third construct used our BclA N-terminal domain to localize the YFP expression to the cell membrane. The microscopy results show that the expression of YFP is nearly continuous around the outside of the cell and that the cell has normal E. colimorphology. It is important to address that in all three images there is 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 internal expression of YFP at the dipoles will not have an impact on our agglutination assay, the cell surface expression is the important part. The first image is E. coli expressing YFP externally using BclA at 100X magnification. The second image is this same image contrast normalized and zoomed in on the E. coli.

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.