Team:LMU-Munich/Spore Coat Proteins/result

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GFP as a Proof of Principle

Finally, we started with the most important experiment for our GFP-Sporobeads, the fluorescence microscopy. For a better understanding of our results, you can see in the table below the five constructs and strain numbers we used in the following experiments.

We developed a sporulation protocol (for details see Protocol for enhancement of mature spore numbers) that increases the rates of mature spores in our samples. The cells were fixed on agarose pads and investigated by phase contrast and fluorescence microscopy. While spores of the wild type only showed the known background fluorescence, all Sporobeads showed bright green fluorescence at the edge (on the surface) of the spores. Sporobeads from strain B 53 (containing the P_cotYZ-cotZ-2aa-gfp-terminator construct) showed the highest fluorescence intensity (see Fig. 5 and all data). Hence, this strain was chosen for further experiments.

Fig. 5: Fluorescence of wild type spores and B53-Sporobeads (containing the PcotYZ-cotZ-gfp-terminator construct)

This two short time lapse videos shows the formation of a spore and lollowing lysis of the mother cell.

Because of the low but distinct fluorescence of wild type spores, we measured and compared the fluorescence intensity of 100 spores per construct (see data). We obtained significant differences between wild type spores and all of our Sporobeads (see data). The following graph (Fig. 6) shows the microscopy pictures and analysis of the strongest of the five constructs integrated into wildtype W168 (B53) and the deletion strain B 49 (B70).

Fig. 6: Result of fluorescence evaluation of the three strains W168, B53 and B70. The bar charts show the average fluorescence intensity over 100-200 spores. For image analysis we measured the fluorescence intensity of an area of 750 pixel per spore by using ImageJ and evaluated the results with the statistical software R. The 3D graphs illustrate in one spore the distribution of fluorescence intensity across the spore surface.

As shown in Fig. 6, the wild type spore has hardly any fluorescence, whereas both Sporobeads with the integrated construct pSB_Bs1C-P_cotYZ-cotZ_-2aa-gfp-terminator give a distinct fluorescence signal around the edge of the spore. Furthermore, it demonstrates that strain B 70 has the highest fluorescence intensity. For more detailed information look at our Data page

In summary we successfully developed functional Sporobeads that are capable of displaying any protein of choice on the surface of modified B. subtilis endospores.