Team:LMU-Munich/Data/Knockout

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Knockouts of germination genes

We induced our germination-mutant strains to sporulate in Difco sporulation media. We quantified the cells and spores per milliliter of each culture using a Neubauer cell counting chamber. Then we plated known dilutions of culture, and measured the germination rate of mutant spores in a germination assay (see Protocols).

The germination rates of B. subtilis germination mutants. All germination rates are relative to wild type 168 (WT168), our positive control. The mutant spo0A::tet is unable to form spores, and should therefore show no germination (our negative control). Mutants used were:
WT168: Wild-type 168
spo0A::tet: A sporulation-deficient strain
B29: cwlD::kan, sleB::mls
B30: gerD::cm, sleB::mls
B32: cwlJ::spec, cwlD::kan
B40: cwlD::kan, sleB::mls, cwlJ::spec
B41: cwlD::kan, sleB::mls, gerD::cm
B42: cwlD::kan, cwlJ::spec, gerD::cm
B43: gerD::cm, sleB::mls, cwlJ::spec
B46: cwlD::kan, cwlJ::spec, gerD::cm, sleB::mls
B47: gerD::cm, 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.

Plates of germination mutants. Plates contained heated (to kill live cells and leave only spores to germinate) cultures and unheated (where colonies may come from germinated spores and the live cells) cultures. The mutant spo0A::tet is unable to form spores, and should therefore show no germination (our negative control). The mistake of photographing empty plates against a background of spots was realized after plates had been discarded. Presence of colonies is noted below, for clarity. Mutants used were:
WT168: Wild-type 168; Heated plate: colonies present; Unheated plate: colonies present
spo0A::tet: A sporulation-deficient strain; Heated plate: no colonies; Unheated plate: colonies present
B42: cwlD::kan, cwlJ::spec, gerD::cm; Heated plate: colonies present; Unheated plate: colonies present
B47: gerD::cm, sleB::mls, cwlJ::spec, cwlD::kan; Heated plate: no colonies; Unheated plate> colonies present

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 ===Knockouts of germination genes ===
-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.
+We induced our germination-mutant strains to sporulate in Difco sporulation media. We quantified the cells and spores per milliliter of each culture using a Neubauer cell counting chamber. Then we plated known dilutions of culture, and measured the germination rate of mutant spores in a germination assay (see [https://2012.igem.org/Team:LMU-Munich/Lab_Notebook/Protocols Protocols]).
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