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Team:LMU-Munich/Data
From 2012.igem.org
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| + | =='''Sporo'''beads== | ||
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| + | ===Spore purification=== | ||
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| + | |<p align="justify">We compared different ways of purifying ''Bacillus'' spores from vegetative cells</p> | ||
| + | |[[File:LMU Purification preview.tiff|200px|link=Team:LMU-Munich/Data/Sporepurification]] | ||
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| + | ! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/Sporepurification]] | ||
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==='''Sporo'''beads=== | ==='''Sporo'''beads=== | ||
Revision as of 17:42, 19 September 2012
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".
[ more news ]
Data
Here you will find all of our project data. For our big breakthroughs, or to follow specific projects, see the individual project pages:
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Bacillus BioBrickBOX |
SporeCoat FusionProteins |
Germination STOP |
BacillusBioBrickBox
Anderson Promoter Evaluation
Evaluation of the Anderson library in B. subtilis with pSBBs3C-lux (luminescence) and pSBBs1C-lacZ | 
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Constitutive Bacillus Promoters
Evaluation of constitutive B. subtilis promoters with pSBBs3C-lux and pSBBs1C-lacZ | 
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Inducible Bacillus Promoters
Evaluation of inducible B. subtilis promoters with pSBBs3C-lux and pSBBs1C-lacZ | 
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Sporobeads
Spore purification
We compared different ways of purifying Bacillus spores from vegetative cells | 
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Sporobeads
To purify our spores from the vegetative cells we treated the samples, that were grown for 24 hours in DS-Medium, with three different methods. The data is summarized in the following table:
| all cells | mature spores | immature spores | |
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| 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 |
Tab. 1 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 the impurity of these samples that 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 microscopy 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.
Germination Stop
We induced our germination-mutant strains to sporulate in Difco sporulation media. Then we measured the germination rate of mutant spores in a germination assay.
Strains:
B40 -- cwlD::kan, sleB::mls, cwlJ::spec
B41 -- cwlD::kan, sleB::mls, gerD::cat
B42 -- cwlD::kan, cwlJ::spec, gerD::cat
B43 -- gerD::cat, sleB::mls, cwlJ::spec
B46 -- cwlD::kan, cwlJ::spec, gerD::cat, sleB::mls
B47 -- gerD::cat, sleb::mls, cwlJ::spec, cwlD::kan
The plate growth demonstrates the inability of our mutant spores to germinate. We can say that fewer than 1 out of 3x10^7 spores of strains B40, B41, B43, B46, and B47 germinated.
