Team:LMU-Munich/Data/Knockout

From 2012.igem.org

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===Knockouts of germination genes ===
===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. Then we measured the germination rate of mutant spores in a germination assay.
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[[File:germination_assay_bahhumbug.jpg|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. <br /> Strains: <br /> '''B40''' -- ''cwlD''::kan, ''sleB''::mls, ''cwlJ''::spec <br /> '''B41''' -- ''cwlD''::kan, ''sleB''::mls, ''gerD''::cat <br /> '''B42''' -- ''cwlD''::kan, ''cwlJ''::spec, ''gerD''::cat <br /> '''B43''' -- ''gerD''::cat, ''sleB''::mls, ''cwlJ''::spec <br /> '''B46''' -- ''cwlD''::kan, ''cwlJ''::spec, ''gerD''::cat, ''sleB''::mls <br /> '''B47''' -- ''gerD''::cat, ''sleb''::mls, ''cwlJ''::spec, ''cwlD''::kan|thumb|400px]]
 
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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.
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.
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<font color="#000000"; size="2">'''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). Mutants used were: <br />
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'''WT168''': Wild-type 168 <br />
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'''''spo0A''::tet''': A sporulation-deficient strain <br />
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'''B42''': ''cwlD''::kan, ''cwlJ''::spec, ''gerD''::cm <br />
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'''B47''': ''gerD''::cm, ''sleB''::mls, ''cwlJ''::spec, ''cwlD''::kan</font>
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Revision as of 21:21, 25 September 2012

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