Team:LMU-Munich/Data

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Revision as of 17:21, 19 September 2012

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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 Intro	Bacillus BioBrickBOX	SporeCoat FusionProteins	Germination STOP

Anderson Promoter Evaluation

Evaluation of the Anderson library in B. subtilis with pSBBs3C-lux (luminescence) and pSBBs1C-lacZ

Constitutive Bacillus" Promoters

Evaluation of constitutive B. subtilis promoters with pSBBs3C-lux and pSBBs1C-lacZ

Inducible Bacillus" Promoters

Evaluation of inducible B. subtilis promoters with pSBBs3C-lux and pSBBs1C-lacZ

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:

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.

Fig. 5: Comparison of colony growth on LB-agar (plus antibiotics) plates. Upper plates are plated with DSM cell/spore cultures that have been heated at 80°C for 1 hour to kill living cells (but does not affect spores). This prevents cells from forming colonies. Therefore, all colonies observed are from germinated spores. Lower plates contain cultures which have not been heated. Growth on these plates is from either cells or spores, and demonstrates that strains are able to grow. WT168 has no germination knockouts; strains B40-B43 have triple knockouts; strains B46 and B47 have quadruple knockouts; Spo0A has a knockout of the sporulation gene Spo0A (replaced with a tet cassette). WT168 is a positive control; Spo0A is a negative control.
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.