Team:LMU-Munich/Spore Coat Proteins/mainresult

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GFP as a Proof of Principle

We designed the first GFP-Sporobead to evaluate our system and compare different constructs.
This short time lapse video shows the formation of a spore.

To ensure that our Sporobeads show fluorescence on their edge and higher than in wildtype spores, we analysed the microscopy pictures. The following graph (Fig. 6) shows these microscopy pictures and analysis of the strongest of the five constructs integrated into wildtype strain (B53) and the deletion strain (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 have later 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.