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Team:LMU-Munich/Germination Stop
From 2012.igem.org
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* Knock out as much genes as possible that are involved in germination | * Knock out as much genes as possible that are involved in germination | ||
| - | * [[ | + | * [[Team:LMU-Munich/Germination_Stop#Suicideswitch|'''Suicide'''switch]]: Toxin production if first fails |
Based on the work of [http://www.ncbi.nlm.nih.gov/pubmed/19554258 J. Kim and W. Schumann (2009)] and [http://www.ncbi.nlm.nih.gov/pubmed/11466293 B. Setlow et al (2001)], we decided to knock out genes ''cwlJ'', ''sleB'', ''cwlB'', ''gerD'', and ''cwlD''. The genes ''cwlJ'' and ''sleB'' code for lytic enzymes which are active in the process of germination. In the work of [http://www.ncbi.nlm.nih.gov/pubmed/11466293 B. Setlow et al (2001)], when ''cwlJ'' and ''sleB'' were knocked out together, germination frequency was reduced by 5 orders of magnitude. [http://www.ncbi.nlm.nih.gov/pubmed/19554258 J. Kim and W. Schumann (2009)] reports a similar reduction in germination when ''gerD'' and ''cwlB'' are knocked out together, and a reduction in germination ability when the gene ''cwlD'' is knocked out. When all five of these genes are knocked out, we hope to yield a ''B. subtilis'' strain which produces spores completely incapable of germination. | Based on the work of [http://www.ncbi.nlm.nih.gov/pubmed/19554258 J. Kim and W. Schumann (2009)] and [http://www.ncbi.nlm.nih.gov/pubmed/11466293 B. Setlow et al (2001)], we decided to knock out genes ''cwlJ'', ''sleB'', ''cwlB'', ''gerD'', and ''cwlD''. The genes ''cwlJ'' and ''sleB'' code for lytic enzymes which are active in the process of germination. In the work of [http://www.ncbi.nlm.nih.gov/pubmed/11466293 B. Setlow et al (2001)], when ''cwlJ'' and ''sleB'' were knocked out together, germination frequency was reduced by 5 orders of magnitude. [http://www.ncbi.nlm.nih.gov/pubmed/19554258 J. Kim and W. Schumann (2009)] reports a similar reduction in germination when ''gerD'' and ''cwlB'' are knocked out together, and a reduction in germination ability when the gene ''cwlD'' is knocked out. When all five of these genes are knocked out, we hope to yield a ''B. subtilis'' strain which produces spores completely incapable of germination. | ||
Revision as of 12:04, 5 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 ]
Germination Stop
The goal of this project is to remove the germination capability, while keeping the necessary structural functions of the spores intact.
There are two approaches to achive this:
- Knock out as much genes as possible that are involved in germination
- Suicideswitch: Toxin production if first fails
Based on the work of [http://www.ncbi.nlm.nih.gov/pubmed/19554258 J. Kim and W. Schumann (2009)] and [http://www.ncbi.nlm.nih.gov/pubmed/11466293 B. Setlow et al (2001)], we decided to knock out genes cwlJ, sleB, cwlB, gerD, and cwlD. The genes cwlJ and sleB code for lytic enzymes which are active in the process of germination. In the work of [http://www.ncbi.nlm.nih.gov/pubmed/11466293 B. Setlow et al (2001)], when cwlJ and sleB were knocked out together, germination frequency was reduced by 5 orders of magnitude. [http://www.ncbi.nlm.nih.gov/pubmed/19554258 J. Kim and W. Schumann (2009)] reports a similar reduction in germination when gerD and cwlB are knocked out together, and a reduction in germination ability when the gene cwlD is knocked out. When all five of these genes are knocked out, we hope to yield a B. subtilis strain which produces spores completely incapable of germination.
Two methods are being employed to knock out germination: resistance cassette (RC) knockouts and clean deletions. Single RC knockouts were created, and have then been combined to create multiple knockouts.
This will be checked with germination assays.
Further, we will investigate the possibility of using a redundant toxin system to immediately kill off any spores which somehow germinate.
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Bacillus BioBrickBOX |
SporeCoat FusionProteins |
Germination STOP |
