"
Team:LMU-Munich/Data/Knockout
From 2012.igem.org
(Difference between revisions)
| Line 10: | Line 10: | ||
[[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]] | [[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]] | ||
| + | |||
| + | {| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;" | ||
| + | | style="width: 70%;background-color: #EBFCE4;" | | ||
| + | {|align:center | ||
| + | |[[File:Germination_RatesII.jpg|600px|center]] | ||
| + | |- | ||
| + | | style="width: 80%;background-color: #EBFCE4;" | | ||
| + | {| style="color:black;" cellpadding="3" width="95%" cellspacing="0" border="0" align="left" style="text-align:left;" | ||
| + | |style="width: 70%;background-color: #EBFCE4;" | | ||
| + | <font color="#000000"; size="2">'''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: <br /> | ||
| + | '''WT168''': Wild-type 168 <br /> | ||
| + | '''''spo0A''::tet''': A sporulation-deficient strain <br /> | ||
| + | '''B29''': ''cwlD''::kan, ''sleB''::mls <br /> | ||
| + | '''B30''': ''gerD''::cm, ''sleB''::mls <br /> | ||
| + | '''B32''': ''cwlJ''::spec, ''cwlD''::kan <br /> | ||
| + | '''B40''': ''cwlD''::kan, ''sleB''::mls, ''cwlJ''::spec <br /> | ||
| + | '''B41''': ''cwlD''::kan, ''sleB''::mls, ''gerD''::cm <br /> | ||
| + | '''B42''': ''cwlD''::kan, ''cwlJ''::spec, ''gerD''::cm <br /> | ||
| + | '''B43''': ''gerD''::cm, ''sleB''::mls, ''cwlJ''::spec <br /> | ||
| + | '''B46''': ''cwlD''::kan, ''cwlJ''::spec, ''gerD''::cm, ''sleB''::mls <br /> | ||
| + | '''B47''': ''gerD''::cm, ''sleB''::mls, ''cwlJ''::spec, ''cwlD''::kan</font> | ||
| + | |} | ||
| + | |} | ||
| + | |} | ||
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. | ||
Revision as of 21:13, 25 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 ]
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.
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.
