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Team:LMU-Munich/Data/Sporepurification
The LMU-Munich team is exuberantly happy about the great success at the World Championship Jamboree in Boston. Our project Beadzillus finished 4th and won the prize for the "Best Wiki" (with Slovenia) and "Best New Application Project".
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Sporobead purification
To be able to use our Sporobeads we have to make sure that all remaining vegetative cells surrounding them are deleted. in oder to purify our spores we treated the samples, that were grown for 24 hours in Difco Sporulation Medium, with three different methods: French Press, sonification and lysozyme. The results and comparison of these three methods is shown in the following table:
| all cells | mature spores | immature spores | |
|---|---|---|---|
| untreated wildtype | 7.29 x 108 /ml | 1 x 108 /ml 13.71% | 0.04 x 108 /ml 0.55% |
| untreated PcotYZ-cotZre-gfp-terminator | 6.79 x 108 /ml | 1 x 108 /ml 14.72% | 0.13 x 108 /ml 1.9% |
| French Press wildtype | 4.87 x 108 /ml | 2.1 x 108 /ml 43% | 0.05 x 108 /ml 1% |
| French Press PcotYZ-cotZre-gfp-terminator | 4.75 x 108 /ml | 1.88 x 108 /ml 39.58% | 0.05 x 108 /ml 1% |
| Sonification wildtype | 4.6 x 108 /ml | 1.22 x 108 /ml 26.52% | 0.1 x 108 /ml 2% |
| Sonification PcotYZ-cotZre-gfp-terminator | 6.72 x 108 /ml | 1.53 x 108 /ml 22.77% | 0.23 x 108 /ml 3% |
| Lysozyme wildtype | 2.48 x 108 /ml | 1.58 x 108 /ml 63.7% | 0 /ml |
| Lysozyme PcotYZ-cotZre-gfp-terminator | 1.05 x 108 /ml | 1.05 x 108/ml 100% | 0 /ml |
The table shows that after treatment with French Press and ultrasound the number of spores compared to the untreated samples was increased. We assume the reason for this was that the samples contained impurities derived from damaged vegetative cells. Thus, during counting it was not always possible to distinguish between mature spores and cell waste. However, a huge difference in number of vegetative cells and spores between untreated and lysozyme treated samples was noticeable under the microscope as it is visualized in the table above and the pictures below. Because the vegetative cells were lyzed and not damaged it was easy to recognize the mature spores. In fig. 1 phase contrast pictures of samples after treatement with French Press, ultrasound and lysozyme are shown.
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The microscopy pictures support the results of fig 1, that the only successful treatment was the one with lysozyme. This is why we tested additionally the fluorescence of Sporobeads treated with it. It revealed that the lysozyme did not harm the fusion protein as fluorescence was detected (see fig 2).
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