Team:LMU-Munich/Spore Coat Proteins

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Sporobeads - What Protein Do You Want to Display?

What are Sporobeads?

Introduction to B. subtilis spores and their assignment for our project

Crust Promoter Evaluation

Three sporulation-dependent promoters (PcotYZ, PcotV, PcgeA) were constructed, two of which show the expected behavior.

Crust Protein Gene Organization

Location of the two crust protein genes in the genome

Cloning Strategy

Cloning strategy to create different variants of our Sporobeads

Cloning Results

Main results of the various constructs we created

But we were able to finish five constructs and integrated them into wild type W168 and the ΔcotZ mutant: </p>

Finally, we started with the most important experiment for our GFP-Sporobeads, the fluorescence microscopy. 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)

Since there were still some vegetative cells left after 24 hours of growth in Difco Sporulation Medium, we wanted to purify the Sporobeads. We tried three different methods for this approach: treatment with French Press, sonification and lysozyme. By means of the microscopy results we were able to conclude that lysozyme treatment was the only successful method (see data). Additionally, this treatment did not harm the crust fusion proteins as green fluorescence was still detectable afterwards (see data for details).

Moreover, we wondered if clean deletions of the native spore crust genes would show any difference in fusion protein expression in our Sporobeads. Thus, we deleted the native cotZ and cgeA using the pMAD based gene deletion strategy described by [http://www.ncbi.nlm.nih.gov/pubmedterm=New%20Vector%20for%20Efficient%20Allelic%20Replacement%20in%20Naturally%20Nontransformable%2C%20Low-GC-Content%2C%20Gram-Positive%20Bacteria Arnaud et al., 2004].

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 intensity bar charts in Fig. 6 show the fluorescence intensity, while the 3D graphs illustrate the distribution of fluorescence intensity across the spore surface. This correlates with the localization of our fusion proteins in the crust. 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 following graph (Fig. 6) shows the results of microscopy and ImageJ analysis of the strongest construct 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.

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.

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

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