Team:LMU-Munich/Germination Stop

From 2012.igem.org

(Difference between revisions)
Line 17: Line 17:
* [[Team:LMU-Munich/Germination_Stop#Suicideswitch|'''Suicide''' switch]]: Toxin production by vegetative cells if germination knockout fails and spores manage to germinate.
* [[Team:LMU-Munich/Germination_Stop#Suicideswitch|'''Suicide''' switch]]: Toxin production by vegetative cells if germination knockout fails and spores manage to germinate.
 +
==How does Germination Work?==
==How does Germination Work?==
 +
 +
[[File:sporulation_diagram.jpg|thumb|Fig. 3: Taken from [http://www.ncbi.nlm.nih.gov/pubmed/19554258 Kim, J. & Schumann W (2009)]. <br />
 +
'''A''': Cell at stage 0; vegetative phase. <br />
 +
'''B''': Cell at stage II; asymmetric septum has been formed. <br />
 +
'''C''': Cell at stage III; cytoplasmic membrane has engulfed forespore. <br />
 +
'''D''': Cell at stage IV; coat formation has started; spore will be released from lysed mother cell.]]
<p align="justify">The ''Bacillus'' life cycle can include both classic division, and also reproduction by sporulation and spore germination.
<p align="justify">The ''Bacillus'' life cycle can include both classic division, and also reproduction by sporulation and spore germination.
In response to starvation of nutrients (including carbon, nitrogen, or phosphorus) or in response to peptides secreted by other cells which signal too high of population densities to cells, ''Bacillus'' cells form spores in a process called sporulation.
In response to starvation of nutrients (including carbon, nitrogen, or phosphorus) or in response to peptides secreted by other cells which signal too high of population densities to cells, ''Bacillus'' cells form spores in a process called sporulation.
The “mother” cell forms the endospore within its own cell membrane. The endospore contains its DNA in the spore core, which is protected by several layers of coats. The outermost layer is the spore crust. The spore is very dry, and contains a substance called dipicolinic acid (DPA), which is replaced with water when the spore germinates. Until the spore hydrates and swells out of its protective coats, it is resistant to a wide variety of environmental stressors, including UV radiation, toxic chemicals, freezing, high heat, dessication, and pH extremes. This resistance to stressors allows the spore to survive until conditions are good for growth.</p>
The “mother” cell forms the endospore within its own cell membrane. The endospore contains its DNA in the spore core, which is protected by several layers of coats. The outermost layer is the spore crust. The spore is very dry, and contains a substance called dipicolinic acid (DPA), which is replaced with water when the spore germinates. Until the spore hydrates and swells out of its protective coats, it is resistant to a wide variety of environmental stressors, including UV radiation, toxic chemicals, freezing, high heat, dessication, and pH extremes. This resistance to stressors allows the spore to survive until conditions are good for growth.</p>
-
 
-
[[File:sporulation_diagram.jpg|Fig. 3: Taken from [http://www.ncbi.nlm.nih.gov/pubmed/19554258 Kim, J. & Schumann W (2009)]. <br />
 
-
A: Vegetative cell in the phase 0. <br />
 
-
B: Cell at stage II where the asymmetric septum has been formed. <br />
 
-
C: Cell at stage III where the cytoplasmic membrane has engulfed the forespore. <br />
 
-
D: Cell at stage IV where formation of the coat has already started; the spore is about to be released from the lysed mother cell.|thumb|368px]]
 
-
 
<p align="justify">On its inner spore membrane, the spore has germinant receptors. The spore coats are believed to be semipermeable or porous, in order to permit the passage of germinants to the receptors. When germinants such as amino acids and sugars reach germinant receptors, the spore begins a biochemical process of germination. It takes up water, shifts its pH, and swells. It breaks out of its coat and begins the outgrowth process. We wish to prevent the germination process.</p>
<p align="justify">On its inner spore membrane, the spore has germinant receptors. The spore coats are believed to be semipermeable or porous, in order to permit the passage of germinants to the receptors. When germinants such as amino acids and sugars reach germinant receptors, the spore begins a biochemical process of germination. It takes up water, shifts its pH, and swells. It breaks out of its coat and begins the outgrowth process. We wish to prevent the germination process.</p>
-
[[File:breaking_out_of_spore_coat.jpg|Fig. 4: From [http://www.photonics.com/Article.aspx?AID=29892 Photonics. com (2007)] Emergence of vegetative cells: 60- to 70-nm-deep apertures in the rodlet later that gradually enlarged (C and D), and subsequently eroded the entire spore coat (E). Germ cells emerged from these apertures. (Photo courtesy Lawrence Livermore National Laboratory)|thumb|341px]]
 
==How do Germination Gene Knockouts Work?==
==How do Germination Gene Knockouts Work?==
<p align="justify">Based on the work of others, we chose to knock out genes ''cwlJ'', ''sleB'', ''cwlB'', ''gerD'', and ''cwlD''. Past works showed:
<p align="justify">Based on the work of others, we chose to knock out genes ''cwlJ'', ''sleB'', ''cwlB'', ''gerD'', and ''cwlD''. Past works showed:
-
*''cwlJ'' and ''sleB'': both genes code for lytic enzymes which are active in the process of germination. When knocked out together, germination frequency was reduced by 5 orders of magnitude. 
+
*'''''cwlJ'' and ''sleB''''': both genes code for lytic enzymes which are active in the process of germination. When knocked out together, germination frequency was reduced by 5 orders of magnitude. 
-
*''gerD'' and ''cwlB'': the ''gerD'' product plays unknown role in nutrient germination; ''cwlB'''s product plays role in cell wall turnover & cell lysis. When knocked out together, germination frequency was reduced by 5 orders of magnitude. 
+
*'''''gerD'' and ''cwlB''''': the ''gerD'' product plays unknown role in nutrient germination; ''cwlB'''s product plays role in cell wall turnover & cell lysis. When knocked out together, germination frequency was reduced by 5 orders of magnitude. 
-
*''cwlD'': this gene codes for recognition components for cleavage by the germination-specific cortex lytic enzymes. When knocked out, germination occurred at a rate of 0.003 to 0.05%.
+
*'''''cwlD''''': this gene codes for recognition components for cleavage by the germination-specific cortex lytic enzymes. When knocked out, germination occurred at a rate of 0.003 to 0.05%.
By knocking out all five of these genes, our goal was to yield a ''B. subtilis'' strain which produces spores completely incapable of germination. The stop to germination comes during the process of spore coat breakdown. Without the lytic enzymes to break down the coat, the spores should be unable to outgrow into the vegetative stage.
By knocking out all five of these genes, our goal was to yield a ''B. subtilis'' strain which produces spores completely incapable of germination. The stop to germination comes during the process of spore coat breakdown. Without the lytic enzymes to break down the coat, the spores should be unable to outgrow into the vegetative stage.
After several attempts to knock out ''cwlB'', and producing extremely slow-growing mutants, ''cwlB'' was removed from our list of genes to knock out.</p>
After several attempts to knock out ''cwlB'', and producing extremely slow-growing mutants, ''cwlB'' was removed from our list of genes to knock out.</p>
Line 66: Line 65:
|0.003 – 0-005%
|0.003 – 0-005%
|}
|}
 +
 +
==What Methods Did We Use to Knockout Germination Genes?==
==What Methods Did We Use to Knockout Germination Genes?==
Line 71: Line 72:
<p align="justify">Two methods were employed to knock out germination: resistance cassette knockouts and clean deletions. Resistance cassette (RC) knockouts were performed using long-flanking-homology PCR (see [https://2012.igem.org/Team:LMU-Munich/Lab_Notebook/Protocols Protocols]). Single RC knockouts were created first; then they were combined to create multiple knockouts.  
<p align="justify">Two methods were employed to knock out germination: resistance cassette knockouts and clean deletions. Resistance cassette (RC) knockouts were performed using long-flanking-homology PCR (see [https://2012.igem.org/Team:LMU-Munich/Lab_Notebook/Protocols Protocols]). Single RC knockouts were created first; then they were combined to create multiple knockouts.  
-
[[File:germination_gene_knockouts_img.jpg|Fig. 1: The four germination genes being knocked out, and their resistance cassette replacements, as shown on the ''Bacillus'' chromosome.|thumb|610px]]
+
[[File:germination_gene_knockouts_circle.jpg|Fig. 1: The four germination genes being knocked out, and their resistance cassette replacements, as shown on the ''Bacillus'' chromosome.|thumb|610px]]
The germination rate of our mutants were checked with a germination assay. The assay was developed based on the protocols of others. For our method, see [https://2012.igem.org/Team:LMU-Munich/Lab_Notebook/Protocols Protocols].
The germination rate of our mutants were checked with a germination assay. The assay was developed based on the protocols of others. For our method, see [https://2012.igem.org/Team:LMU-Munich/Lab_Notebook/Protocols Protocols].

Revision as of 12:04, 25 September 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

Team-LMU streaked plate.resized.jpg

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".

IGEM HQ LMU prize.jpg

[ more news ]

Sporenfreunde

Retrieved from "http://2012.igem.org/Team:LMU-Munich/Germination_Stop"