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	== '''Germination'''STOP ==		== '''Germination'''STOP ==
-	<p align="justify">The goal of this subproject was to yield '''Sporo'''beads which are safe (not able to germinate) and consistently functional (maintain their spore shape and structure). To achieve this, we sought to remove the germination capability of our spores, while keeping their necessary structural functions intact.</p>	+	<p align="justify">The goal of this subproject was to yield Sporobeads which are safe (unable to germinate) and consistently functional (maintain their spore shape and structure). To achieve this, we sought to remove the germination capability of our spores, while keeping their necessary structural functions intact.</p>
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	* [[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?==
-		+	<p align="justify">The ''Bacillus'' life cycle can include both classic division, and also reproduction by sporulation and spore germination.
-	==Gene Knockouts==	+	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>
-	<p align="justify">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)] report 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.</p>	+
-		+
-	<p align="justify">Two methods are being employed to knock out germination: resistance cassette (RC) knockouts and clean deletions. Single RC knockouts were created first; then they were combined to create multiple knockouts.</p>	+
-		+
-	[[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:LFH_image.jpg|Fig. 2: The technique employed for resistance cassette knockouts: Long-flanking-homology PCR. This example shows the gene ''cwlD'' being replaced with the resistance cassette kanamycin.|thumb|610px]]	+
-		+
-	This will be checked with germination assays.	+
-		+
-	<p align="justify">Further, we will investigate the possibility of using a redundant toxin system to immediately kill off any spores which somehow germinate in the '''Suicide'''switch system.</p>	+
-		+
-		+
-	====How do the Gene Knockouts of '''Germination'''STOP actually work?====	+
-		+
-	<p align="justify">To understand how our germination knockouts actually prevent germination, it is crucial to understand how the ''Bacillus'' germination process occurs.</p>	+
-		+
-	<p align="justify">''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. </p>	+
-	<p align="justify">The “mother” cell forms the endospore within its own cell membrane. The spore contains its DNA in the spore core, which is protected by several layers of coats. 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 is hydrated (and swells), it is resistant to a wide variety of environmental stressors, including UV radiation, toxic chemicals, freezing, high heat, dessication, and pH extremes.</p>	+
	[[File:sporulation_diagram.jpg|Fig. 3: Taken from [http://www.ncbi.nlm.nih.gov/pubmed/19554258 Kim, J. & Schumann W (2009)]. <br />		[[File:sporulation_diagram.jpg|Fig. 3: Taken from [http://www.ncbi.nlm.nih.gov/pubmed/19554258 Kim, J. & Schumann W (2009)]. <br />
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	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]]		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">The spore has germinant receptors on its inner spore membrane, and is suspected to have semipermeable or porous outer layers that 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.</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]]		[[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]]
-	<p align="justify">We were concerned that because the beginning stage of germination is a strictly biochemical one, that maybe our spores would lose the ability to fully germinate, but would nonetheless become deformed by the initial steps of germination. This would be problematic because the spores should be vectors to carry proteins; deformed spores could be ineffective as delivery vectors. The investigative work of [http://www.ncbi.nlm.nih.gov/pubmed/17535925 Plomp et al (2007)] on ''Bacillus'' cells seems to support that the lytic enzyme knockouts we chose could help to maintain the spore shape. They state:</p>	+	==How do Germination Gene Knockouts Work?==
-		+
-	::“A significant fraction (≈30%) of spores did not proceed to outgrowth in the timeframe of the observation and did not exhibit degradation of the rodlet layer. However, after drying, >90% of these spores showed a structural collapse, indicating prior replacement of the dipicolinic acid in the spore core with water, i.e. they did proceed through the germination stage, but not the outgrowth stage.” […] “Etch pits were the initiation sites for early germination-induced spore coat fissure formation.” […] “Disassembly of the higher-order rodlet structure initiates at micro-etch pits, and proceeds by the expansion of the pits to form fissures perpendicular to the rodlet direction.” […] “We suggest by analogy that rodlet structure degradation is caused by specific hydrolytic enzyme(s), located within the spore integument and activated during the early stages of germination.”	+
-		+
-	<p align="justify">What we read from this is that spores can be cued to germinate, and the etch pits that lead to the destruction of the spore coat can be formed, but without the activity of lytic enzymes, further steps of germination do not occur. Therefore, it is possible for our spores to be presented with germinants, and still to maintain their structural shape, despite possibly undergoing the first biochemical stage of germination.</p>	+
-		+
		+	<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.
		+	*''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%.
		+	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>
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	|}		|}
		+	==What Methods Did We Use to Knockout Germination Genes?==
		+
		+	<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]]
		+
		+	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].
-	<p align="justify">Thus, by knocking out the genes coding for lytic enzymes, our goal is to prevent both outgrowth, and the disassembly of the spore coat. This will leave our '''Sporo'''Beads intact to be delivery vectors for proteins.</p>	+	In the event that some small percentage of spores retained the ability to germinate, the '''Suicide''' switch subproject (below) prevents outgrowth into viable vegetative cells.</p>

---

Revision as of 11:52, 25 September 2012

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GerminationSTOP

The goal of this subproject was to yield Sporobeads which are safe (unable to germinate) and consistently functional (maintain their spore shape and structure). To achieve this, we sought to remove the germination capability of our spores, while keeping their necessary structural functions intact.

Two approaches were used to achieve this:

• Knock out genes that are involved in germination.

• Suicideswitch: Toxin production by vegetative cells if germination knockout fails and spores manage to germinate.

How does Germination Work?

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. 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.

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.

How do Germination Gene Knockouts Work?

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. 
• 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%.

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.

Germination Genes	Gene Function	Mutant Germination Rate
gerD	Unknown role in nutrition germination	Reduction by 5 orders of magnitude
cwlJ	Germination-specific lytic enzymes	0.003 – 0.05% No ATP detected
sleB	Germination-specific lytic enzymes	0.003 – 0.05% No ATP detected
cwlD	Recognition component for lytic enzymes	0.003 – 0-005%

What Methods Did We Use to Knockout Germination Genes?

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 Protocols). Single RC knockouts were created first; then they were combined to create multiple knockouts.

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 Protocols.

In the event that some small percentage of spores retained the ability to germinate, the Suicide switch subproject (below) prevents outgrowth into viable vegetative cells.

Suicideswitch

Genetic elements of the suicide switch and its expected performance.

As a backup plan to make our Sporobeads even safer, we developed the Suicideswitch. In case the spores do germinate, due to degradation or destruction of their outer coats, e.g. by high pressure, the Suicideswitch will be turned on.

It is composed by an alternative sigma factor ecf41, which derives from B. lincheniformes and we chose to work with the short version constitutively on. It is synthetically linked to a sigma G regulated promotor responding quite late to sigma G (PspoIVB responding strongly or PsspK responding weakly) which is the last sigma factor activated in the forespore. Through this ecf41 is produced quite late in the forespore. Ecf41 then activates the PydfG promotor, which is the naturally responding promoter to ecf41, which then activates the transcription of MazF, a bacterial toxin from E.coli degrading mRNA.

The idea behind this is to pack the Sporobeads full with ecf41 when they sporulate, which will in turn kill them upon germination due to the MazF.

Through the system with the alternative sigma factor the forespore gains hopefully enough time to fully mature before MazF starts to be produced.

We chose MazF, as we think it will not be able to harm our Sporobead even if it is made too early, as the spore does not rely on translation to be preserved.

We are planning to model this system.