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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 two short time lapse videos show the formation of a spore and following lysis of the mother cell of one of our constructs.

We had to ensure that production is both high and takes place on the edge of spores. 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.