Team:LMU-Munich/Germination Stop

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== '''Germination'''STOP ==
 
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== '''Germination'''STOP ==
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<p align="justify">The goal of this module was to yield [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads] which are safe (unable to germinate) and consistently functional (maintain their spore shape and structure throughout time). 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 module was to yield [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads] which are safe (unable to germinate) and consistently functional (maintain their spore shape and structure throughout time). 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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[[File:GerminationSTOP_cycleII.jpg|620px|center]]
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[[File:GerminationSTOP_cycleIIii.jpg|620px|center]]
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Two approaches were used to achieve this:
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[[File:NEXT.png|right|80px|link=Team:LMU-Munich/safetytour]] [[File:BACK.png|left|80px|link=Team:LMU-Munich/Bacillus_BioBricks]]
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* [[Team:LMU-Munich/Germination_Stop#How do Germination Gene Knockouts Work?|Knock out]] genes that are involved in germination.
 
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* [[Team:LMU-Munich/Germination_Stop#Suicide switch|'''Suicide''' switch]]: Toxin production by vegetative cells if germination knockout fails and spores manage to germinate.
 
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==How do Sporulation & Germination Work?==
 
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|[[File:sporulation_diagram.jpg|200px]]
 
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<font color="#000000"; size="2">Fig. 1: Taken from [http://www.ncbi.nlm.nih.gov/pubmed/19554258 Kim, J. & Schumann W (2009)]. <br />
 
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'''A''': Cell at stage 0; vegetative phase. <br />
 
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'''B''': Cell at stage II; asymmetric septum has been formed. <br />
 
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'''C''': Cell at stage III; cytoplasmic membrane has engulfed forespore. <br />
 
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'''D''': Cell at stage IV; coat formation has started; spore will be released from lysed mother cell.</font>
 
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<p align="justify">The ''Bacillus'' life cycle can include both classic division as well as reproduction by sporulation and spore germination.
 
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In response to starvation of nutrients (including carbon, nitrogen, or phosphorus) or in response to peptides secreted by other cells which signal high population densities, ''Bacillus'' cells form spores in a process called sporulation (Fig. 1).
 
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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>
 
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<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 the receptors, the spore begins the biochemical process of germination (see Fig. 2).
 
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During germination, the spore replaces its DPA with water (Stages I and II in Fig. 2), shifts its pH (Stage I), and swells (Stage II). Lytic enzymes break down the rigid spore coat layers encapsulating the spore's core (Stage II), allowing the spore core to swell further and begin the outgrowth process into a vegetative cell (Outgrowth). When the spore coat is broken down by lytic enzymes, the spore's environmental resistance is lost. For our project, we wish to prevent the germination process.</p>
 
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|[[File:germination_from_spore.jpg|620px|center]]
 
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<font color="#000000"; size="2">Fig. 2: '''The germination process in ''Bacillus'' spores.''' Taken from [http://www.ncbi.nlm.nih.gov/pubmed/14662349 Setlow 2003]. </font>
 
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==How do Germination Gene Knockouts Work?==
 
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<p align="justify">Our idea of how to prevent germination from occurring is to knock out genes involved in the germination process. There are a huge number of genes which play a role in germination, but our goal was to select a few very essential genes to knock out.</p>
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<p align="justify">Based on the research of others, the genes we chose were ''cwlJ'', ''sleB'', ''cwlB'', ''gerD'', and ''cwlD''. Past works 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)] showed:</p>
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==Gene Knockouts==
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*'''''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. 
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{| "width=100%" style="text-align:center;" style="align:right"|
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*'''''gerD'' and ''cwlB''''': the ''gerD'' product plays an unknown role in nutrient germination; the product of ''cwlB'' plays a role in cell wall turnover & cell lysis. When knocked out together, germination frequency was reduced by 5 orders of magnitude. Note: after several attempts to knock out ''cwlB'', and yielding cells unable to grow or cells growing at a very slow rate, we determined that knocking out ''cwlB'' would not be prudent for the production of [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''beads], as it would delay all experiments in the '''Germination'''STOP module.
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|<p align="justify">We picked important germination genes and knocked them out. Subsequently we combined the single mutants into quadruple mutants and successfully prevented germination.</p>  
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*'''''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%.
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|[[File:LMU Germination STop plate.png|200px|right|link=Team:LMU-Munich/Germination_Stop/Knockout]]
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<p align="justify">By knocking out all five of these genes, our goal was to yield a ''B. subtilis'' strain which produces spores completely incapable of germination. Germination is stopped 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.
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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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{| class="colored"
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!Germination Genes
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!Gene Function
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!Mutant Germination Rate
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!''gerD''
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Germination_Stop/Knockout]]
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|Unknown role in nutrition germination
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|Reduction by 5 orders of magnitude
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!''cwlJ''
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|Germination-specific lytic enzymes
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|0.003 – 0.05%
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No ATP detected
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!''sleB''
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|Germination-specific lytic enzymes
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|0.003 – 0.05%
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No ATP detected
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!''cwlD''
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|Recognition component for lytic enzymes
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|0.003 – 0-005%
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==Germination Gene Knockout Success!==
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<p align="justify">Two methods were employed to knock out germination genes: replacement by resistance cassettes 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. A graphical representation of the genome with all replacements with resistance cassettes can be seen in Fig 3.</p>
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<font color="#000000"; size="2">Fig. 3: Schematic representation of the B. subtilis chromosome with four germination genes replaced by different resistance cassettes. For more information about these knockout strains, please see our [https://2012.igem.org/Team:LMU-Munich/Strains Strain Collection].</font>
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<p align="justify">The germination rate of our mutants were checked with a [[Team:LMU-Munich/Lab_Notebook/Protocols|germination assay]]. The assay was developed based on the protocols of other researchers.
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<br></p>
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<p align="justify">We were able to achieve germination rates as low as '''<1 in 4.6x10<sup>9</sup>''' spores in our triple mutant B43. Other mutants also showed extremely reduced germination ability. A table of germination rates of our mutants and all other results can be found on [https://2012.igem.org/Team:LMU-Munich/Data/Knockout our results page]. Our single mutants were not checked in the germination assay. The double and triple mutants tested showed mixed results, with some unable to germinate, and others maintaining germination ability. Our quadruple mutants demonstrated no ability to germinate, even several separate germination assays.
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<br></p>
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<p align="justify">Despite this success, safety was a top priority for this project. 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>
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=='''Suicide'''switch==
=='''Suicide'''switch==
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|<p align="justify">In case of germination gene knockout failure, we invented the suicide switch. If spores still germinate, the production of a toxin leads to immediate cell death.</p>
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{| style="color:black;" cellpadding="3" width="70%" cellspacing="0" border="0" align="center" style="text-align:left;"
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|[[File:LMU SuicideSwitch grafik.png|200px|right|link=Team:LMU-Munich/Germination_Stop/SuicideSwitch]]
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|[[File:LMU SuicideSwitch grafik.png|624px|center]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Germination_Stop/SuicideSwitch]]
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<font color="#000000"; size="2">Fig. 4: Genetic elements of the '''Suicide''' switch and its expected performance.</font>
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<p align="justify">As a backup plan to make our [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins <b>Sporo</b>beads] even safer, we developed the <b>Suicide</b>switch. In case the spores do germinate, due to degradation or destruction of their outer coats, e.g. by high pressure, the <b>Suicide</b>switch will be turned on.<br></p>  
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<p align="justify">We take advantage of an alternative and heterologous σ factor and the target promoter. Both are not present in ''B. subtilis'' and therefore not recognized by any other σ factor of ''B. subtilis''.</p>
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<p align="justify">The alternative sigma factor [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823043 ecf41<sub>Bli aa1-204</sub>] ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426412/ Wecke T. et al 2012]), which derives from ''B. licheniformis'' a truncated version constitutively "ON" is synthetically linked to one out of two σ<sup>G</sup>-regulated promoters ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K823048 P<sub>''spoIVB''</sub>] ([http://www.ncbi.nlm.nih.gov/pubmed/15699190 Steil L. et al. 2005]) responding strongly or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823042 P<sub>sspK</sub>] ([http://www.ncbi.nlm.nih.gov/pubmed/15699190 Steil L., Völker U. et al. 2005]) responding weakly). σ<sup>G</sup> is the last σ factor activated in the forespore. Consequently, Ecf41<sub>Bli aa1-204</sub> is produced quite late and only in the forespore. Ecf41<sub>Bli aa1-204</sub> then activates its unique target promoter [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823041 P<sub>''ydfG''</sub>] ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426412/ Wecke T., Mascher T. 2012]). This promoter is fused to [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823044 MazF] ([http://jcs.biologists.org/content/118/19/4327.abstract Engelberg-Kulka H.,Amitai S. 2005]). Activation of P<sub>''ydfG''</sub> therefore leads to the expression of MazF, a bacterial toxin from ''E.coli'' degrading mRNA. <br></p>
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<p align="justify">The resulting time course of expression is as follows: Sporulation of the [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins <b>Sporo</b>beads] leads to Ecf41<sub>Bli aa1-204</sub> production in the forespore at the end of the differentiation cycle, through the activation of either P<sub>''spoIVB''</sub> or P<sub>sspK</sub>. Subsequently, this would lead to the expression of MazF, due to the activation of P<sub>''ydfG''</sub>. For our already matured [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins <b>Sporo</b>beads], this would not be toxic, as they represent a dormant state that does not require translation any more. But in the case that a [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''bead] escapes the '''Germination'''STOP based on the gene knockouts described above, a germinating cell will already contain or soon start producing MazF and therefore kill the vegetative cells.<br></p>
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See what [https://2012.igem.org/Team:LMU-Munich/Data/Suicideswitch Data] we got when measuring a module with ''luxABCDE'' instead of MazF.
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We are planning to model this system, but still need more quantitative, time-resolved data for it. We hope to get this data after finishing the cloning of P<sub>''sspK''</sub> and MazF into ''B. subtilis''.</p>
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