Template:Team:Bonn:NetworkData

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content.i = 2;

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content.titleShort = "Protein regulation";  

content.titleShort = "Protein regulation";  

content.titleLong = "Protein regulation mechanisms";  

content.titleLong = "Protein regulation mechanisms";  

content.i = 4;

content.i = 4;

content.childs=[];

content.childs=[];

content.titleShort = "Protein cleavage";

content.titleShort = "Protein cleavage";

content.i = 5;  

content.i = 5;  

content.childs=[];

content.childs=[];

content.titleShort = "Gene knock-out and knock-in";

content.titleShort = "Gene knock-out and knock-in";

content.titleLong = "Direct inhibition and activation";

content.titleLong = "Direct inhibition and activation";

content.summary= "Direct binding of small molecules in order to activate or inactivate protein function";

content.summary= "Direct binding of small molecules in order to activate or inactivate protein function";

content.type="Background";

content.type="Background";

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content.titleLong = "Ec. SspB adaptor";

content.titleLong = "Ec. SspB adaptor";

content.summary= "The sspB protein is an adaptor responsible for delivering ssrA-tagged substrates to the ClpXP protease in order to enhance their degradation";

content.summary= "The sspB protein is an adaptor responsible for delivering ssrA-tagged substrates to the ClpXP protease in order to enhance their degradation";

content.type="Background";

content.type="Background";

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content.summary= "The LOV-ipaA -vinculin system is a combined system for light inducible heterodimerisation. This powerful tool, which allows photocontroled complex formation was establish by Lungu et al. in 2012.";

content.summary= "The LOV-ipaA -vinculin system is a combined system for light inducible heterodimerisation. This powerful tool, which allows photocontroled complex formation was establish by Lungu et al. in 2012.";

content.text= "The LOV-ipaA -vinculin system is a combined system for light inducible heterodimerisation. It consists out of a LOV domain, which undergoes conformational changes upon irradiation with blue light, and the ipaA-vinculin hybridization system. This two building blocks have be combined and described by Lungu et al. in 2012.</br></br> Lungu et al. (2008) where able to measure a 49-fold difference in target binding upon irradiation as compared to the dark state. However, they further modified the system by mutations of the LOV-ipaA construct and successfully weakend the baseline affinity for vinculin (initial design: 3.5 nM to 69 nM; mutant: 2.4 nM to >40µM affinity for vinculin) to reduce the dark state activity. </br></br> Lov-ipaA-VinD1 is a powerful tool which allows photocontroled complex formation. To establish this system Lungu et al. (2012)^{<a href=#361>36.1</a>} fused the Ja helix of the LOV Domain to ipaA.</br>To be more precise they used the LOV2 domain from Avena sativa photopropin 1 (AsLOV2), which – as previously shown – can be used to photomodulate the affinity of peptides for their binding partners (see Figure 1). </br><div class=contant-image><img src=https://static.igem.org/mediawiki/2013/0/02/Bonn_MS_Figure1_LOV.jpg></br>Figure 1: General design of AsLOV2 fusion proteins (Lungu et al. 2012)^{<a href=#361>36.1</a>}</div> </br>In other studies had been shown that the LOV domain can be fused to entire protein domains, allowing photomodulation of the protein binding. However, they stated that it might be of high importance to bring the LOV domain closer to ipaA, in order to allow photomodulation in this case, because ipaA is only a peptide and thus more flexible than folded domains.</br></br>Therefore, Lungu et al. (2012)^{<a href=361>36.1</a>} identified similar amino acid sequences in the ipaA peptide and the Ja helix of the LOV Domain and used this combined with molecular modeling to create photomodulateable AsLOV2-ipaA (see Figure 2). </br><div class=contant-image><img src=https://static.igem.org/mediawiki/2013/5/54/Bonn_MS_Figure2_LOV-ipaA.jpg></br>Figure 2: Light-inducible LOV-ipaA construct (Lungu et al. 2012)^{<a href=361>36.1</a>}</div></br>They were able to proof the functionality of the AsLOV2-ipaA system by heterodimerization in yeast (yeast two-hybrid system or Y2H) The yeast two-hybrid system can be used to monitor protein–protein interactions between two proteins. The system is based on a transcription factor, which is split into two separate fragments, called the binding domain (BD) and activating domain (AD). Each domain is fused to one protein and thus only if the proteins interact with each other BD and AD are close enough to initiate the transcription of a reporter gene.</br></br>The basic principle of the LOV-ipaA & VinD1 system works as follows. In the dark state the fusion product LOV-ipaA is not able to form a complex with vinculin, because LOV blocks ipaA sterically. However, activation of the LOV domain with blue light induces conformational changes in the fused molecule, which results in a movement of the Ja helix with the ipaA away from LOV. Thereby, ipaA becomes accessible for VinD1 and a Complex is formed.</br></br>The activation is reversible and the entire system can be genetically encoded. This two facts are the main advantages of this system in contrast to other typically used systems, which like for the chemical system for example, are based on in vivo covalently modified peptides, that can be activated by light induced cleavage. Moreover, the protein used are relatively small and thus should interfere as little as possible with the prokaryotic metabolism, the activity change form dark to light state is high, the system is completely genetically encoded and reversible. But, the most important property of this system is that it allows the light-controlled heterodimerisation of the two split variants of sspB, which is necessary for our system.</br></br><h2>References</h2></br><a name=361>36.1</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334866/>Lungu et al. (2012) Designing photoswitchable peptides using the AsLOV2 domain</a>";

content.text= "The LOV-ipaA -vinculin system is a combined system for light inducible heterodimerisation. It consists out of a LOV domain, which undergoes conformational changes upon irradiation with blue light, and the ipaA-vinculin hybridization system. This two building blocks have be combined and described by Lungu et al. in 2012.</br></br> Lungu et al. (2008) where able to measure a 49-fold difference in target binding upon irradiation as compared to the dark state. However, they further modified the system by mutations of the LOV-ipaA construct and successfully weakend the baseline affinity for vinculin (initial design: 3.5 nM to 69 nM; mutant: 2.4 nM to >40µM affinity for vinculin) to reduce the dark state activity. </br></br> Lov-ipaA-VinD1 is a powerful tool which allows photocontroled complex formation. To establish this system Lungu et al. (2012)^{<a href=#361>36.1</a>} fused the Ja helix of the LOV Domain to ipaA.</br>To be more precise they used the LOV2 domain from Avena sativa photopropin 1 (AsLOV2), which – as previously shown – can be used to photomodulate the affinity of peptides for their binding partners (see Figure 1). </br><div class=contant-image><img src=https://static.igem.org/mediawiki/2013/0/02/Bonn_MS_Figure1_LOV.jpg></br>Figure 1: General design of AsLOV2 fusion proteins (Lungu et al. 2012)^{<a href=#361>36.1</a>}</div> </br>In other studies had been shown that the LOV domain can be fused to entire protein domains, allowing photomodulation of the protein binding. However, they stated that it might be of high importance to bring the LOV domain closer to ipaA, in order to allow photomodulation in this case, because ipaA is only a peptide and thus more flexible than folded domains.</br></br>Therefore, Lungu et al. (2012)^{<a href=361>36.1</a>} identified similar amino acid sequences in the ipaA peptide and the Ja helix of the LOV Domain and used this combined with molecular modeling to create photomodulateable AsLOV2-ipaA (see Figure 2). </br><div class=contant-image><img src=https://static.igem.org/mediawiki/2013/5/54/Bonn_MS_Figure2_LOV-ipaA.jpg></br>Figure 2: Light-inducible LOV-ipaA construct (Lungu et al. 2012)^{<a href=361>36.1</a>}</div></br>They were able to proof the functionality of the AsLOV2-ipaA system by heterodimerization in yeast (yeast two-hybrid system or Y2H) The yeast two-hybrid system can be used to monitor protein–protein interactions between two proteins. The system is based on a transcription factor, which is split into two separate fragments, called the binding domain (BD) and activating domain (AD). Each domain is fused to one protein and thus only if the proteins interact with each other BD and AD are close enough to initiate the transcription of a reporter gene.</br></br>The basic principle of the LOV-ipaA & VinD1 system works as follows. In the dark state the fusion product LOV-ipaA is not able to form a complex with vinculin, because LOV blocks ipaA sterically. However, activation of the LOV domain with blue light induces conformational changes in the fused molecule, which results in a movement of the Ja helix with the ipaA away from LOV. Thereby, ipaA becomes accessible for VinD1 and a Complex is formed.</br></br>The activation is reversible and the entire system can be genetically encoded. This two facts are the main advantages of this system in contrast to other typically used systems, which like for the chemical system for example, are based on in vivo covalently modified peptides, that can be activated by light induced cleavage. Moreover, the protein used are relatively small and thus should interfere as little as possible with the prokaryotic metabolism, the activity change form dark to light state is high, the system is completely genetically encoded and reversible. But, the most important property of this system is that it allows the light-controlled heterodimerisation of the two split variants of sspB, which is necessary for our system.</br></br><h2>References</h2></br><a name=361>36.1</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334866/>Lungu et al. (2012) Designing photoswitchable peptides using the AsLOV2 domain</a>";

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content.titleShort = "Project summary";  

content.titleShort = "Project summary";  

content.titleLong = "Our project in a nutshell";

content.titleLong = "Our project in a nutshell";

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content.childs=[41,74,43];  

content.childs=[41,74,43];  

content.titleShort = "C. crescentus";

content.titleShort = "C. crescentus";

content.text= "Caulobacter crescentus is a Gram-negative &alpha;-protobacterium often found in fresh water lakes or in the sea. Its cell division cycle is a favoured object of study due to its remarkable asymmetry. ^{<a href=401>40.1</a>}</br>ClpXP is a protease that degrades proteins tagged with the ssrA peptide. sspB&alpha; is an adaptor protein that can accelerate protein degradation by tethering ssrA-tagged proteins towards the ClpX subunit of the ClpXP protease.</br>For further information, please browse the related articles. </br></br><h2>References</h2></br><a href=401>40.1</a><a href=’ http://www.amazon.com/Brock-Biology-Microorganisms-13th-Edition/dp/032164963X’> Brock Microbiology of Microorganisms, Madigan et al., Pearson, German edition Vol. 13, 2012</a>";

content.text= "Caulobacter crescentus is a Gram-negative &alpha;-protobacterium often found in fresh water lakes or in the sea. Its cell division cycle is a favoured object of study due to its remarkable asymmetry. ^{<a href=401>40.1</a>}</br>ClpXP is a protease that degrades proteins tagged with the ssrA peptide. sspB&alpha; is an adaptor protein that can accelerate protein degradation by tethering ssrA-tagged proteins towards the ClpX subunit of the ClpXP protease.</br>For further information, please browse the related articles. </br></br><h2>References</h2></br><a href=401>40.1</a><a href=’ http://www.amazon.com/Brock-Biology-Microorganisms-13th-Edition/dp/032164963X’> Brock Microbiology of Microorganisms, Madigan et al., Pearson, German edition Vol. 13, 2012</a>";

content.type="Project";

content.type="Project";

content.parents=[40];  

content.parents=[40];  

content.childs=[];  

content.childs=[];  

content.summary= "This article is about the ClpXP protease system in C. crescentus and regulation of proteolysis via ClpXP. It also provides information about similarities and differences between C. crescentus and E. coli orthologs of related proteins.";

content.summary= "This article is about the ClpXP protease system in C. crescentus and regulation of proteolysis via ClpXP. It also provides information about similarities and differences between C. crescentus and E. coli orthologs of related proteins.";

content.text= "<div class='content-image'><img src='https://static.igem.org/mediawiki/2013/d/de/Bonn-41-CC-ClpXP.png'>ClpXP mediated proteolysis ^{<a href=#413>41.3</a>}</div>ClpXP is an AAA+ protease which is of particular importance for proper cell-cycle progression. It consists of a hexameric ClpX subunit, which recognizes and unfolds tagged proteins, while ATP is hydrolyzed, and a ClpP subunit, that contains the actual peptidase domain. ^{<a href=#411>41.1</a>, <a href=#412>41.2</a>} Its structure is highly conserved, such that E. coli and C. crescentus orthologs are very similar. </br>Specific activity of the ClpXP protease is mediated by sspB and ssrA. ssrA is a short peptide consisting of fourteen amino acids in C. crescentus. Proteins which need to be degraded, e.g. for regulatory purpose or due to errors in their structure, are tagged with the ssrA peptide that can be recognized by the ClpX subunit. sspB is a dimeric adaptor protein in C. crescentus which tethers ssrA-tagged proteins to the ClpXP protease and, in this way, accelerates protein degradation. </br>Whilst sspB protein structure is well- conserved among many microorganisms, the structure of ^CCsspB&alpha; (i.e., the C. crescentus ortholog of sspB) shows significant differences compared to other orthologs. For example, ^CCsspB&alpha; and ^ECsspB (i.e., the E. coli ortholog) only show up with sequence identities of 16%, while CCsspB is still able to specifically bind to ^ECClpXP^{<a href=#412>41.2</a>}. SsrA tags can also be very different among microorganismic species. </br>This makes it possible to establish a protein degradation system in E. coli involving ^CCsspB&alpha; and ^ECClpXP, as long as the ssrA tag can be recognized by both ^CCsspB&alpha; and ^ECClpXP. </br></br><h2>References</h2></br><a name=411>41.1</a> <a href=’ http://www.ncbi.nlm.nih.gov/pubmed/17937918’>Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a></br><a name=412>41.2</a> <a href=’http://www.ncbi.nlm.nih.gov/pubmed/20014030’>Versatile modes of peptide recognition by the ClpX N domain mediate alternative adaptor-binding specificities in different bacterial species, Chowdhury et al., Protein Science, 2010, PMID: 20014030</a></br><a name=413>41.3</a> <a href='http://www.biochem.umass.edu/faculty/peter-chien'>Peter Chien, Department of Biochemistry and Molecular Biology (official website), http://www.biochem.umass.edu/faculty/peter-chien, picture URL: cchttp://www.biochem.umass.edu/sites/biochem/files/resize/ClpX-300x268.jpg</a>";

content.text= "<div class='content-image'><img src='https://static.igem.org/mediawiki/2013/d/de/Bonn-41-CC-ClpXP.png'>ClpXP mediated proteolysis ^{<a href=#413>41.3</a>}</div>ClpXP is an AAA+ protease which is of particular importance for proper cell-cycle progression. It consists of a hexameric ClpX subunit, which recognizes and unfolds tagged proteins, while ATP is hydrolyzed, and a ClpP subunit, that contains the actual peptidase domain. ^{<a href=#411>41.1</a>, <a href=#412>41.2</a>} Its structure is highly conserved, such that E. coli and C. crescentus orthologs are very similar. </br>Specific activity of the ClpXP protease is mediated by sspB and ssrA. ssrA is a short peptide consisting of fourteen amino acids in C. crescentus. Proteins which need to be degraded, e.g. for regulatory purpose or due to errors in their structure, are tagged with the ssrA peptide that can be recognized by the ClpX subunit. sspB is a dimeric adaptor protein in C. crescentus which tethers ssrA-tagged proteins to the ClpXP protease and, in this way, accelerates protein degradation. </br>Whilst sspB protein structure is well- conserved among many microorganisms, the structure of ^CCsspB&alpha; (i.e., the C. crescentus ortholog of sspB) shows significant differences compared to other orthologs. For example, ^CCsspB&alpha; and ^ECsspB (i.e., the E. coli ortholog) only show up with sequence identities of 16%, while CCsspB is still able to specifically bind to ^ECClpXP^{<a href=#412>41.2</a>}. SsrA tags can also be very different among microorganismic species. </br>This makes it possible to establish a protein degradation system in E. coli involving ^CCsspB&alpha; and ^ECClpXP, as long as the ssrA tag can be recognized by both ^CCsspB&alpha; and ^ECClpXP. </br></br><h2>References</h2></br><a name=411>41.1</a> <a href=’ http://www.ncbi.nlm.nih.gov/pubmed/17937918’>Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a></br><a name=412>41.2</a> <a href=’http://www.ncbi.nlm.nih.gov/pubmed/20014030’>Versatile modes of peptide recognition by the ClpX N domain mediate alternative adaptor-binding specificities in different bacterial species, Chowdhury et al., Protein Science, 2010, PMID: 20014030</a></br><a name=413>41.3</a> <a href='http://www.biochem.umass.edu/faculty/peter-chien'>Peter Chien, Department of Biochemistry and Molecular Biology (official website), http://www.biochem.umass.edu/faculty/peter-chien, picture URL: cchttp://www.biochem.umass.edu/sites/biochem/files/resize/ClpX-300x268.jpg</a>";

content.parents=[74];  

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content.summary= "This article deals with the structure of sspB&alpha; and conformational details of its binding to ssrA and ClpXP during tethering.";

content.summary= "This article deals with the structure of sspB&alpha; and conformational details of its binding to ssrA and ClpXP during tethering.";

content.text= "<div class='content-image'><img src='https://static.igem.org/mediawiki/2013/thumb/4/42/Bonn_OutlookCCSspB1_Version2.png/726px-Bonn_OutlookCCSspB1_Version2.png'>sspB structure and its conservation among C. crescentus, E. coli and H. influenzae ^{<a href=#421>42.1</a>}</div>The sspB&alpha; dimeric structure is stabilized by two &alpha;-helices in interaction, as part B of the figure above shows, each of them located at the N-terminus of either sspB&alpha; molecule. The subsequent parts of the protein form a domain consisting of two &beta;-sheet structures, together building up the ssrA binding site. An unstructured area at the C-terminus being referred to as the XB module forms the ClpX binding part of the protein. It is connected to the rest of the molecule via a linker domain. ^{<a href=#422>42.2</a>} </br>Chien et al. ^{<a href=#421>42.1</a>} compared crystal structures of C. crescentus sspB&alpha; and its E. coli and H. influenzae sspB orthologs, discovering that in sspB&alpha; the &alpha;-helices are significantly longer, more twisted and cover a larger cross section area than the other two sspB orthologs. Also considering that &beta;-sheets are rotated by around 20&deg; in comparison to E. coli and H. influenzae orthologs, this leads to an antiparallel orientation of the two ssrA tagged protein bound to the ssrA binding sites of an sspB&alpha; dimer in C. crescentus, while they are parallel in &gamma;-protobacterial sspB.</br></br><div class='content-image'><img src='https://static.igem.org/mediawiki/2013/6/6c/Bonn_OutlookCCSspB2.png' align=left>By measuring GFP fluorescence intensity, decrease of GFP-^CCssrA concentration (1) without sspB&alpha; added, (2) with mutated sspB&alpha;(Q74A) added , (3) with wildtype sspB&alpha; added can be visualized. ^{<a href=#421>42.1</a>}</div>Chien et al. point out that although there are the remarkable differences in protein structure between sspB&alpha; and its &gamma;-protobacterial ortholog, they show up with similar effectiveness in binding proteins tagged with the related ssrA peptide. But it turned out in their research that effectiveness of sspB&alpha; binding to the protein which needs to be tethered to the ClpXP protease strongly depends on which ssrA ortholog the protein is tagged with. sspB&alpha; binds firmly to ^CCssrA, with an affinity being 175 times as large as for binding to ^ECssrA (i.e. the E. coli ortholog). By comparing the crystal structures of both sspB&alpha; and the compound of sspB&alpha; and ^CCssrA, Chien et al. further proved that binding of sspB&alpha; to ^CCssrA does not lead to significant changes of its 3D conformation.</br></br><h2>References</h2></br><a name=421>42.1</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/17937918'>Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a></br><a name=422>42.2</a><a href='http://www.ncbi.nlm.nih.gov/pubmed/14967151'> Bivalent tethering of sspB to ClpXP is required for efficient substrate delivery: a protein design study, Bolon DN et al., Mol Cell, 2004, PMID: 14967151</a>";

content.text= "<div class='content-image'><img src='https://static.igem.org/mediawiki/2013/thumb/4/42/Bonn_OutlookCCSspB1_Version2.png/726px-Bonn_OutlookCCSspB1_Version2.png'>sspB structure and its conservation among C. crescentus, E. coli and H. influenzae ^{<a href=#421>42.1</a>}</div>The sspB&alpha; dimeric structure is stabilized by two &alpha;-helices in interaction, as part B of the figure above shows, each of them located at the N-terminus of either sspB&alpha; molecule. The subsequent parts of the protein form a domain consisting of two &beta;-sheet structures, together building up the ssrA binding site. An unstructured area at the C-terminus being referred to as the XB module forms the ClpX binding part of the protein. It is connected to the rest of the molecule via a linker domain. ^{<a href=#422>42.2</a>} </br>Chien et al. ^{<a href=#421>42.1</a>} compared crystal structures of C. crescentus sspB&alpha; and its E. coli and H. influenzae sspB orthologs, discovering that in sspB&alpha; the &alpha;-helices are significantly longer, more twisted and cover a larger cross section area than the other two sspB orthologs. Also considering that &beta;-sheets are rotated by around 20&deg; in comparison to E. coli and H. influenzae orthologs, this leads to an antiparallel orientation of the two ssrA tagged protein bound to the ssrA binding sites of an sspB&alpha; dimer in C. crescentus, while they are parallel in &gamma;-protobacterial sspB.</br></br><div class='content-image'><img src='https://static.igem.org/mediawiki/2013/6/6c/Bonn_OutlookCCSspB2.png' align=left>By measuring GFP fluorescence intensity, decrease of GFP-^CCssrA concentration (1) without sspB&alpha; added, (2) with mutated sspB&alpha;(Q74A) added , (3) with wildtype sspB&alpha; added can be visualized. ^{<a href=#421>42.1</a>}</div>Chien et al. point out that although there are the remarkable differences in protein structure between sspB&alpha; and its &gamma;-protobacterial ortholog, they show up with similar effectiveness in binding proteins tagged with the related ssrA peptide. But it turned out in their research that effectiveness of sspB&alpha; binding to the protein which needs to be tethered to the ClpXP protease strongly depends on which ssrA ortholog the protein is tagged with. sspB&alpha; binds firmly to ^CCssrA, with an affinity being 175 times as large as for binding to ^ECssrA (i.e. the E. coli ortholog). By comparing the crystal structures of both sspB&alpha; and the compound of sspB&alpha; and ^CCssrA, Chien et al. further proved that binding of sspB&alpha; to ^CCssrA does not lead to significant changes of its 3D conformation.</br></br><h2>References</h2></br><a name=421>42.1</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/17937918'>Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a></br><a name=422>42.2</a><a href='http://www.ncbi.nlm.nih.gov/pubmed/14967151'> Bivalent tethering of sspB to ClpXP is required for efficient substrate delivery: a protein design study, Bolon DN et al., Mol Cell, 2004, PMID: 14967151</a>";

content.parents=[40];  

content.parents=[40];  

content.childs=[];  

content.childs=[];  

content.summary= "The protein degradation system in Caulobacter crescentus resembles the system in E. coli, but the respective sequences of ssrA and SspB differ. Thus the different specifities can be used to introduce the ccSspB split system in wildtyp E. coli without disturbing the native processes in it.";  

content.summary= "The protein degradation system in Caulobacter crescentus resembles the system in E. coli, but the respective sequences of ssrA and SspB differ. Thus the different specifities can be used to introduce the ccSspB split system in wildtyp E. coli without disturbing the native processes in it.";  

content.text= "The protein degradation system in Caulobacter crescentus resembles the system in E. coli, but the respective sequences of ssrA and SspB differ ^{<a href=#431>43.1</a>}. Thus ccssrA only binds ccSspB but not E. coli SspB. ^{<a href=#432>43.2</a>} ^{<a href=#433>43.3</a>} ^{<a href=#434>43.4</a>} However, proteins tagged with ccssrA can be degraded by E. coli ClpXP. Therefore the utilization of ccSspB and ccssrA in E. coli has the advantage that SspB+ strains can be used. ^{<a href=#431>43.1</a>} </br> In order to use this with the SspB split system, the fusion proteins ccSspBΔ10-FRB and FKBP12-SspB[XB] (E. coli) were incubated with GFP-ccDAS+4 and E. coli ClpXP in vitro. Without rapamycin there was no degradation detected. Equally, addition of E. coli SspB showed no degradation.  Addition of rapamycin led to a reduction of GFP-ccDAS+4 of around 12% in 180 seconds. Compared to the E. coli split system (around 30 % in 180 seconds) this system is less fast but can, at least in vitro, be used with sspB-wildtype E. coli ^{<a href=#431>43.1</a>}. </br> As the results of the E.coli and the C. crescentus system in vitro show many similarities and the E. coli system works in vivo. It may be possible to use the C. crescentus in vivo as well.  </br> <img src='https://static.igem.org/mediawiki/2013/8/82/Bonn-ccSspB.jpg'> ^{<a href=#431>43.1</a>} <h2>References:</h2>  </br> <a name=431>43.1</a> <a href='http://dspace.mit.edu/bitstream/handle/1721.1/58089/654116495.pdf?sequence=1'> Understanding and Harnessing Energy-Dependent Proteolysis for Controlled Protein Degradation in Bacteria, J. Davis, Massachusetts Institute of Technology, april 2010  </a> </br> <a name=432>43.2</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC58509/'> Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis, Flynn et al, Proc Natl Acad Sci USA 2001 Sep 11, PMID: 11535833 </a> </br> <a name=433>43.3</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/17937918'> Structure and substrate specifity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al, Structure 2007 Oct, PMID: 17937918 <a/> </br> <a name=434>43.4</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581644/'> Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP, Griffith and Grossman, Mol Microbiol. 2008 Nov, PMID: 18811726 <a/> </br>";  

content.text= "The protein degradation system in Caulobacter crescentus resembles the system in E. coli, but the respective sequences of ssrA and SspB differ ^{<a href=#431>43.1</a>}. Thus ccssrA only binds ccSspB but not E. coli SspB. ^{<a href=#432>43.2</a>} ^{<a href=#433>43.3</a>} ^{<a href=#434>43.4</a>} However, proteins tagged with ccssrA can be degraded by E. coli ClpXP. Therefore the utilization of ccSspB and ccssrA in E. coli has the advantage that SspB+ strains can be used. ^{<a href=#431>43.1</a>} </br> In order to use this with the SspB split system, the fusion proteins ccSspBΔ10-FRB and FKBP12-SspB[XB] (E. coli) were incubated with GFP-ccDAS+4 and E. coli ClpXP in vitro. Without rapamycin there was no degradation detected. Equally, addition of E. coli SspB showed no degradation.  Addition of rapamycin led to a reduction of GFP-ccDAS+4 of around 12% in 180 seconds. Compared to the E. coli split system (around 30 % in 180 seconds) this system is less fast but can, at least in vitro, be used with sspB-wildtype E. coli ^{<a href=#431>43.1</a>}. </br> As the results of the E.coli and the C. crescentus system in vitro show many similarities and the E. coli system works in vivo. It may be possible to use the C. crescentus in vivo as well.  </br> <img src='https://static.igem.org/mediawiki/2013/8/82/Bonn-ccSspB.jpg'> ^{<a href=#431>43.1</a>} <h2>References:</h2>  </br> <a name=431>43.1</a> <a href='http://dspace.mit.edu/bitstream/handle/1721.1/58089/654116495.pdf?sequence=1'> Understanding and Harnessing Energy-Dependent Proteolysis for Controlled Protein Degradation in Bacteria, J. Davis, Massachusetts Institute of Technology, april 2010  </a> </br> <a name=432>43.2</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC58509/'> Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis, Flynn et al, Proc Natl Acad Sci USA 2001 Sep 11, PMID: 11535833 </a> </br> <a name=433>43.3</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/17937918'> Structure and substrate specifity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al, Structure 2007 Oct, PMID: 17937918 <a/> </br> <a name=434>43.4</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581644/'> Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP, Griffith and Grossman, Mol Microbiol. 2008 Nov, PMID: 18811726 <a/> </br>";  

content.parents=[74];

content.parents=[74];

content.childs=[];

content.childs=[];

content.titleLong = "C. crescentus ssrA and its application in E. coli";

content.titleLong = "C. crescentus ssrA and its application in E. coli";

content.summary= "Proteins that need to be degraded by the ClpXP protease have to be tagged with the ssrA peptide previosly. Here is some information on the structure of ssrA.";

content.summary= "Proteins that need to be degraded by the ClpXP protease have to be tagged with the ssrA peptide previosly. Here is some information on the structure of ssrA.";

content.i =52;

content.i =52;

content.parents=[37];

content.parents=[37];

content.titleShort="Achievements";

content.titleShort="Achievements";

content.titleLong="Achievements";

content.titleLong="Achievements";

content.childs=[67, 68];

content.childs=[67, 68];

content.titleShort = "Kill-switch for Lab Safety";

content.titleShort = "Kill-switch for Lab Safety";

content.summary= "Toxin-antitoxin systems are composed by an antitoxin and a toxin coding gene. Connecting our light inducible protein degradation system to the antitoxin via an ssrA-tag allows light induced cell death, as predominance of the toxin in a bacterium activates a cell death pathway.";

content.summary= "Toxin-antitoxin systems are composed by an antitoxin and a toxin coding gene. Connecting our light inducible protein degradation system to the antitoxin via an ssrA-tag allows light induced cell death, as predominance of the toxin in a bacterium activates a cell death pathway.";

content.text= "Our system of light inducible protein degradation can be utilized to degrade any specific protein and is thus usable in a light induced kill-switch system. For this application a connection between the degradation system and a toxin-antitoxin module like MazEF or ccdA/ccdB is needed. Either the toxin or the antitoxin could be light inducibly degraded by adding an ssrA-tag, which is detected by our degradation system, to its genetical code: <ul><li><b>Using the degradation of the toxin:</b> For that purpose the insertion of a plasmid containing the ssrA-tagged toxin encoding gene is needed. Since the predominance of the toxin activates a cell death pathway in bacteria, a bacterium containing a module that allows light inducible degradation of the toxin would only be viable, when the toxin is degraded. In darkness the toxin overexpression is no longer compensated and aggregation of it leads to cell death. Apart from the use in lab security such a kill-switch system would also be useful for environmental applications of bacteria, since it opens up the possibility of deploying bacteria for only one day and ensures their passing by nightfall. For example bacteria could be used to perform the ecological stabilization of a lake but after one night any genetically modified bacteria would be dead.</li><li><b>Using the degradation of the antitoxin:</b> Two plasmids are needed: The first one to express the toxin and the second one to express the ssra-tagged antitoxin, in such manner that the amounts of the toxin and the antitoxin are in equilibrium. Once light induces the degradation system, the antitoxin is degraded and the predominant toxin will kill the bacterium. </li></ul> Regarding our idea to improve lab security by implementing a kill-switch system, both described ways seem possible. Usage of the former would require cultivating and working with the bacteria under constant blue light, as darkness would kill them. Realization of the latter would require no usage of any blue light in the lab since bacteria which get into touch with daylight our any blue light would be killed. Due to the high light sensivity of our degradation system it can most likely be induced by daylight, which renders the former killswitch system useless. Bacteria which escape from the lab could survive simply through contact with daylight. Consequently we focused on the second system (the degradation of the antitoxin).</br> With MazEF we described a light inducible kill-switch system via the insertion of plasmids into bacteria. However a final kill-switch system would have to be introduced into the genomic DNA since plasmids in bacteria can be ejected, for instance via cell division, whereas a genomic DNA mutation is less likely to occur. Nevertheless the risk of a loss of function cannot be eliminated , which is why a secure system should countain much more than one kill-switch system to compensate the malfunction of a single kill-switch system. Therefore, we consider the MazEF kill-switch system to be part of a much larger security system for genetically engineered bacteria. </br>";

content.text= "Our system of light inducible protein degradation can be utilized to degrade any specific protein and is thus usable in a light induced kill-switch system. For this application a connection between the degradation system and a toxin-antitoxin module like MazEF or ccdA/ccdB is needed. Either the toxin or the antitoxin could be light inducibly degraded by adding an ssrA-tag, which is detected by our degradation system, to its genetical code: <ul><li><b>Using the degradation of the toxin:</b> For that purpose the insertion of a plasmid containing the ssrA-tagged toxin encoding gene is needed. Since the predominance of the toxin activates a cell death pathway in bacteria, a bacterium containing a module that allows light inducible degradation of the toxin would only be viable, when the toxin is degraded. In darkness the toxin overexpression is no longer compensated and aggregation of it leads to cell death. Apart from the use in lab security such a kill-switch system would also be useful for environmental applications of bacteria, since it opens up the possibility of deploying bacteria for only one day and ensures their passing by nightfall. For example bacteria could be used to perform the ecological stabilization of a lake but after one night any genetically modified bacteria would be dead.</li><li><b>Using the degradation of the antitoxin:</b> Two plasmids are needed: The first one to express the toxin and the second one to express the ssra-tagged antitoxin, in such manner that the amounts of the toxin and the antitoxin are in equilibrium. Once light induces the degradation system, the antitoxin is degraded and the predominant toxin will kill the bacterium. </li></ul> Regarding our idea to improve lab security by implementing a kill-switch system, both described ways seem possible. Usage of the former would require cultivating and working with the bacteria under constant blue light, as darkness would kill them. Realization of the latter would require no usage of any blue light in the lab since bacteria which get into touch with daylight our any blue light would be killed. Due to the high light sensivity of our degradation system it can most likely be induced by daylight, which renders the former killswitch system useless. Bacteria which escape from the lab could survive simply through contact with daylight. Consequently we focused on the second system (the degradation of the antitoxin).</br> With MazEF we described a light inducible kill-switch system via the insertion of plasmids into bacteria. However a final kill-switch system would have to be introduced into the genomic DNA since plasmids in bacteria can be ejected, for instance via cell division, whereas a genomic DNA mutation is less likely to occur. Nevertheless the risk of a loss of function cannot be eliminated , which is why a secure system should countain much more than one kill-switch system to compensate the malfunction of a single kill-switch system. Therefore, we consider the MazEF kill-switch system to be part of a much larger security system for genetically engineered bacteria. </br>";

content.i =57;

content.i =57;

content.parents=[37];

content.parents=[37];

content.titleShort = "Results";

content.titleShort = "Results";

content.titleLong = "Results";

content.titleLong = "Results";

content.summary= "Results";

content.summary= "Results";

content.text="";

content.text="";

content.type="Project";

content.type="Project";

content.parents=[37];

content.parents=[37];

content.childs=[];

content.childs=[];

content.summary= "A list of each and every plasmid we designed combined with short summaries of their function";

content.summary= "A list of each and every plasmid we designed combined with short summaries of their function";

content.type="Project";

content.type="Project";

break;

break;

content.childs=[];

content.childs=[];

content.titleShort = "MazEF";

content.titleShort = "MazEF";

content.summary= "The toxin-antitoxin system MazEF is composed by an upstream gene <i>mazE</i>, encoding a labile antitoxin, and a downstream gene <i>mazF</i>, that encodes a stable toxin. Connecting our light inducible protein degradation system to the antitoxin MazE allows light inducible cell death, as predominance of MazF in a bacterium activates cell death pathway.";

content.summary= "The toxin-antitoxin system MazEF is composed by an upstream gene <i>mazE</i>, encoding a labile antitoxin, and a downstream gene <i>mazF</i>, that encodes a stable toxin. Connecting our light inducible protein degradation system to the antitoxin MazE allows light inducible cell death, as predominance of MazF in a bacterium activates cell death pathway.";

content.text= "Our system of light inducible protein degradation can be utilized to degrade any specific protein and is therefore usable to realize a light inducible kill-switch system. A connection of protein degradation to a cell death pathway is represented by the stress-induced toxin-antitoxin module <i>mazEF</i> in Escherichia coli. <i>mazEF</i> is located on the chromosome in E.coli which is associated with programmed cell death. The toxin-antitoxin system is composed by an upstream gene <i>mazE</i>, encoding a labile antitoxin, and a downstream gene mazF, that encodes a stable toxin. </br>The product of <i>mazF</i> cleaves mRNAs and tmRNAs at a specific site, which leads to an inhibition of translation. MazF shows a specific cleaving mechanism, which is not well understood yet, but shows that there is also protein synthesis which is unaffected by MazF. These proteins are presumably part of a cell death pathway. </br>The effect of <i>mazF</i> is suppressed by the Product of <i>mazE</i> which is degraded by the Protease ClpAP in bacteria. As a result of stressful conditions expression of the chromosomal <i>mazEF</i> module is reduced which leads to an imbalance between the products of <i>mazF</i> and <i>mazE</i>: When expression is lowered the stable toxin of <i>mazF</i> still persists while the labile antitoxin of <i>mazE</i> is degraded and can no longer suppress the effect of <i>mazF</i> leading to acute toxicity and cell death. </br><i>MazEF</i>-mediated cell death in E. coli can be caused by:<ul><li>extreme amino acid starvation^{<a href = #674>67.4</a>}^{<a href = #675>67.5</a>}</li><li> inhibition of transcription and/or translation by antibiotics such as rifampin, chloramphenicol, and spectinomycin under specific growth conditions^{<a href = #676>67.6</a>}</li><li>inhibition of translation by the Doc protein of prophage P1^{<a href = #677>67.7</a>}</li><li>DNA damage caused by thymine starvation^{<a href = #678>67.8</a>} as well as by mitomycin C, nalidixic acid, and UV irradiation^{<a href = #679>67.9</a>}</li><li>oxidative stress (H2O2)^{<a href = #679'>67.9</a>}</li></ul>Amitai et al. tested in 2004 the Hypothesis of Pedersen et al.^{<a href = #672>67.2</a>}, that chromosomal toxin-antitoxin systems may rather cause a state of reversible bacteriostasis than programmed cell death^{<a href = #671>67.1</a>}.Therefore E.coli strain MC4100 &#916<i>mazE</i>F relA1 lacIq was cotransformated with:<ul><li>pBad-<i>mazF</i></li><li>pQE-&#916his-<i>mazE</i></li></ul><i>mazF</i>-expression can be induced by the addition of Arabinose via the pBad promoter of the first plasmid. The transformation of the second plasmid results firstly in the repression of <i>mazE</i> expression, whereas when IPTG is added <i>mazE</i> production is induced.</br><div class='content-image' align='center' height=501 width=410><a href='https://static.igem.org/mediawiki/2013/a/ac/Team_Bonn_MazF_1.png'><img src='https://static.igem.org/mediawiki/2013/a/ac/Team_Bonn_MazF_1.png' height=491 width=400></a></br><i>Ability of E. coli cells that had been ectopically overexpressing MazF in liquid medium to form colonies when ectopically overexpressing MazE on plates. The cultures were grown in LB medium (A) or M9 minimal medium with 0.5% glycerol (B) at 37°C to midlogarithmic phase (OD600, 0.5)^{<a href = #671>67.1</a>}.</i></div>Using these tools, Amitai et al. tested the effect of MazE overproduction on MazF-overproducing bacteria during growth in liquid medium.</br>The E.coli strain was incubated in LB medium. After <i>mazF</i> expression was induced by adding arabinose two samples were taken at several time points. To repress <i>mazF</i> expression to both of them glucose was added. In addition IPTG was added to one culture to induce <i>mazE</i> expression. The two cultures were compared via the level of protein synthesis and OD600.</br>Finally Amitai et al. confirmed the assumption that the overproduction of MazE after until 6h under overproduction of MazF could resuscitate E.coli cells in LB medium (Fig. 1A)^{<a href = #671>67.1</a>}, but the longer MazF was induced the less cells could be resuscitated by MazE.</br>Whereas MazE overproduction can reverse the inhibitory effect of MazF on translation, it cannot reverse the effect of MazF on colony formation, which is shown in figure 2. Only 1h after the induction of MazE expression, the rate of translation was restored to nearly 100% (Fig.2 Aa, Ab, Ac) but the bacteriocidic effect could not be reversed (Fig.2 Ba, Bb, Bc).</br>Additionally, Amtai et al. found out, that in M9 medium MazE was less able to reverse the effects of MazF overexpression than in LB medium (Fig.1B vs. 1A). it was concluded that there is a point of no return, when MazE is inable to resuscitate a MazF damaged cell, which occurs earlier in M9 medium than in LB medium.</br>Based on their results a model of the MazEF mechanism was built: A <i>mazF</i>-mediated cascade leads to a cell death pathway, but can nevertheless be stopped at several intermediary steps by e.g. <i>mazE</i>. When a point of no return is reached, the cascade cannot be stopped anymore.<div class='content-image' align='center' height=827 width=784><a href='https://static.igem.org/mediawiki/2013/9/9e/Team_Bonn_MazF_2.png'><img src='https://static.igem.org/mediawiki/2013/9/9e/Team_Bonn_MazF_2.png' height=817 width=764></a></br><i>Effect of MazE overproduction during growth in liquid medium on the ability of MazF-overproducing E. coli cells to synthesize proteins and form colonies. To induce <i>mazE</i> expression, IPTG was added to the bacterial culture at 1 h (Aa and Ba), 4 h (Ab and Bb), and 6 h (Ac and Bc) after <i>mazF</i> induction at time zero. The effects of the ectopic overexpression of MazE were measured at 1 and 3 h after the induction of <i>mazE</i> expression.^{<a href = #671>67.1</a>}.</i></div></br>Back to our project and to the idea of a light inducible kill-switch system:</br>As we described in the previous paragraph for both of our kill-switch systems using the MazEF module either MazF or MazE could be degraded:<ul><li>Using the degradation of MazF:</b> For that purpose the insertion of a plasmid containing the ssrA-tagged toxin encoding gene is needed. Since the predominance of the toxin activates a cell death pathway in bacteria, a bacterium containing a module that allows light inducible degradation of the MazF would only be viable, when it is degraded. In darkness the MazF overexpression is no longer compensated and aggregation of it leads to cell death. </li><li><b>Using the degradation of the antitoxin:</b> Two plasmids are needed: The first one to express the MazF and the second one to express the ssra-tagged MazE, in such manner that the amounts of the <i>MazF</i> and the <i>MazE</i> are in equilibrium. Once light induces the degradation system, the MazE is degraded and the predominant toxin will kill the bacterium.</li></ul>As we explained in the general kill-switch system text we finally focused on the second system (via the degradation of MazE).</br>With the design of a MazEF kill-switch system the possibility of resuscitating bacteria in the way Amitai et al. showed has to be considered. A predominant MazF could kill a bacterium in LB within about two hours, but it needs to be predominant over a long period (>7h) to induce its death without it being resuscitated by renewed <i>mazE</i> expression (Fig.1A)with more than 50% probability .</br>Certainly these facts seem to be unfavourable for the realization of a kill switch system via MazEF, but fortunately our system of heterodimerization (Lungu et al.) allows long continuous degradation^{<a href = #673>67.3</a>}, due to the high stability of the light induced hererodimer. Therefore likely a short exposure time will result in prolonged protein degradation sufficing for bacterial death. Additionally, Amitai et al. showed that the less nutrition is available for a bacterium, the earlier the point of no return is reached. If a bacterium escapes the lab, it will likely have less nutrition available than in LB medium. It might reach the point of no return earlier.</br>We described a light inducible MazEF kill-switch system via the insertion of plasmids into bacteria. However a final kill-switch system would have to be implemented in the genomic DNA since plasmids in bacteria can be ejected, for instance via cell division, whereas a genomic DNA mutation is less likely to occur. Nevertheless risk of a loss of function cannot be eliminated , which is why a secure system should countain much more than one kill-switch system to compensate the malfunction of a single kill-switch system. Therefore, we consider the MazEF kill-switch system to be part of a much larger security system for genetically engineered bacteria.<h2>References</h2><a name =671>67.1</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC532418/'>MazF-Mediated Cell Death in Escherichia coli: a Point of No Return, Shahar Amitai et al., Journal of Bacteriology Vol. 186, No. 24, 2004, p.8295–8300.</a></br><a name =672>67.2</a> <a href = 'http://www.ncbi.nlm.nih.gov/pubmed/?term=Rapid+induction+and+reversal+of+bacteriostatic+conditions+by+controlled+expression+of+toxins+and+antitoxins'>Rapid induction and reversal of bacteriostatic conditions by controlled expression of toxins and antitoxins, Pedersen et al., Molecular Microbiology 45, 2002, 501–510.</a></br><a name =673>67.3</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334866/'>Designing Photoswitchable Peptides Using the AsLOV2 Domain, Oana I. Lungu et al., Chem Biol. 2012, 19(4):507-17.</a></br><a name =674>67.4</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC39188/'>An Escherichia coli chromosomal addiction module regulated by guanosine 3,5-bispyrophosphate: a model for programmed bacterial cell death, Aizenman, E., H. Engelberg-Kulka, and G. Glaser, Proc. Natl. Acad. Sci., 1996, USA 93:6059-6063.</a></br><a name =675>67.5</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC28068/'>rexB of bacteriophage lambda is an anti-cell death gene. Engelberg-Kulka, H., M. Reches, S. Narasimhan, R. Schoulaker-Schwarz, Y. Klemes, E. Aizenman, and G. Glaser, Proc. Natl. Acad. Sci., 1998, USA 95:15481-15486.</a></br><a name =676>67.6</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC95100/'>Programmed cell death in Escherichia coli: some antibiotics can trigger mazEF lethality, Sat, B., R. Hazan, T. Fisher, H. Khaner, G. Glaser, and H. Engelberg-Kulka, J. Bacteriol. 2001, 183:2041-2045.</a></br><a name =677>67.7</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC95101/'>Postsegregational killing mediated by the P1 phage addiction module Phd-Doc requires the Escherichia coli programmed cell death system mazEF, Hazan, R., B. Sat, M. Reches, and H. Engelberg-Kulka, J. Bacteriol. 2001, 183:2046–2050.</a></br><a name =678>67.8</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC150121/'>The Escherichia coli mazEF suicide module mediates thymineless death, Sat, B., M. Reches, and H. Engelberg-Kulka, J. Bacteriol. 2003, 185:1803–1807.</a></br><a name =679>67.9</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC415763/'>Escherichia coli mazEFmediated cell death is triggered by various stressful conditions, Hazan, R., B. Sat, and H. Engelberg-Kulka, J. Bacteriol. 2004, 186:3663–3669.</a>";

content.text= "Our system of light inducible protein degradation can be utilized to degrade any specific protein and is therefore usable to realize a light inducible kill-switch system. A connection of protein degradation to a cell death pathway is represented by the stress-induced toxin-antitoxin module <i>mazEF</i> in Escherichia coli. <i>mazEF</i> is located on the chromosome in E.coli which is associated with programmed cell death. The toxin-antitoxin system is composed by an upstream gene <i>mazE</i>, encoding a labile antitoxin, and a downstream gene mazF, that encodes a stable toxin. </br>The product of <i>mazF</i> cleaves mRNAs and tmRNAs at a specific site, which leads to an inhibition of translation. MazF shows a specific cleaving mechanism, which is not well understood yet, but shows that there is also protein synthesis which is unaffected by MazF. These proteins are presumably part of a cell death pathway. </br>The effect of <i>mazF</i> is suppressed by the Product of <i>mazE</i> which is degraded by the Protease ClpAP in bacteria. As a result of stressful conditions expression of the chromosomal <i>mazEF</i> module is reduced which leads to an imbalance between the products of <i>mazF</i> and <i>mazE</i>: When expression is lowered the stable toxin of <i>mazF</i> still persists while the labile antitoxin of <i>mazE</i> is degraded and can no longer suppress the effect of <i>mazF</i> leading to acute toxicity and cell death. </br><i>MazEF</i>-mediated cell death in E. coli can be caused by:<ul><li>extreme amino acid starvation^{<a href = #674>67.4</a>}^{<a href = #675>67.5</a>}</li><li> inhibition of transcription and/or translation by antibiotics such as rifampin, chloramphenicol, and spectinomycin under specific growth conditions^{<a href = #676>67.6</a>}</li><li>inhibition of translation by the Doc protein of prophage P1^{<a href = #677>67.7</a>}</li><li>DNA damage caused by thymine starvation^{<a href = #678>67.8</a>} as well as by mitomycin C, nalidixic acid, and UV irradiation^{<a href = #679>67.9</a>}</li><li>oxidative stress (H2O2)^{<a href = #679'>67.9</a>}</li></ul>Amitai et al. tested in 2004 the Hypothesis of Pedersen et al.^{<a href = #672>67.2</a>}, that chromosomal toxin-antitoxin systems may rather cause a state of reversible bacteriostasis than programmed cell death^{<a href = #671>67.1</a>}.Therefore E.coli strain MC4100 &#916<i>mazE</i>F relA1 lacIq was cotransformated with:<ul><li>pBad-<i>mazF</i></li><li>pQE-&#916his-<i>mazE</i></li></ul><i>mazF</i>-expression can be induced by the addition of Arabinose via the pBad promoter of the first plasmid. The transformation of the second plasmid results firstly in the repression of <i>mazE</i> expression, whereas when IPTG is added <i>mazE</i> production is induced.</br><div class='content-image' align='center' height=501 width=410><a href='https://static.igem.org/mediawiki/2013/a/ac/Team_Bonn_MazF_1.png'><img src='https://static.igem.org/mediawiki/2013/a/ac/Team_Bonn_MazF_1.png' height=491 width=400></a></br><i>Ability of E. coli cells that had been ectopically overexpressing MazF in liquid medium to form colonies when ectopically overexpressing MazE on plates. The cultures were grown in LB medium (A) or M9 minimal medium with 0.5% glycerol (B) at 37°C to midlogarithmic phase (OD600, 0.5)^{<a href = #671>67.1</a>}.</i></div>Using these tools, Amitai et al. tested the effect of MazE overproduction on MazF-overproducing bacteria during growth in liquid medium.</br>The E.coli strain was incubated in LB medium. After <i>mazF</i> expression was induced by adding arabinose two samples were taken at several time points. To repress <i>mazF</i> expression to both of them glucose was added. In addition IPTG was added to one culture to induce <i>mazE</i> expression. The two cultures were compared via the level of protein synthesis and OD600.</br>Finally Amitai et al. confirmed the assumption that the overproduction of MazE after until 6h under overproduction of MazF could resuscitate E.coli cells in LB medium (Fig. 1A)^{<a href = #671>67.1</a>}, but the longer MazF was induced the less cells could be resuscitated by MazE.</br>Whereas MazE overproduction can reverse the inhibitory effect of MazF on translation, it cannot reverse the effect of MazF on colony formation, which is shown in figure 2. Only 1h after the induction of MazE expression, the rate of translation was restored to nearly 100% (Fig.2 Aa, Ab, Ac) but the bacteriocidic effect could not be reversed (Fig.2 Ba, Bb, Bc).</br>Additionally, Amtai et al. found out, that in M9 medium MazE was less able to reverse the effects of MazF overexpression than in LB medium (Fig.1B vs. 1A). it was concluded that there is a point of no return, when MazE is inable to resuscitate a MazF damaged cell, which occurs earlier in M9 medium than in LB medium.</br>Based on their results a model of the MazEF mechanism was built: A <i>mazF</i>-mediated cascade leads to a cell death pathway, but can nevertheless be stopped at several intermediary steps by e.g. <i>mazE</i>. When a point of no return is reached, the cascade cannot be stopped anymore.<div class='content-image' align='center' height=827 width=784><a href='https://static.igem.org/mediawiki/2013/9/9e/Team_Bonn_MazF_2.png'><img src='https://static.igem.org/mediawiki/2013/9/9e/Team_Bonn_MazF_2.png' height=817 width=764></a></br><i>Effect of MazE overproduction during growth in liquid medium on the ability of MazF-overproducing E. coli cells to synthesize proteins and form colonies. To induce <i>mazE</i> expression, IPTG was added to the bacterial culture at 1 h (Aa and Ba), 4 h (Ab and Bb), and 6 h (Ac and Bc) after <i>mazF</i> induction at time zero. The effects of the ectopic overexpression of MazE were measured at 1 and 3 h after the induction of <i>mazE</i> expression.^{<a href = #671>67.1</a>}.</i></div></br>Back to our project and to the idea of a light inducible kill-switch system:</br>As we described in the previous paragraph for both of our kill-switch systems using the MazEF module either MazF or MazE could be degraded:<ul><li>Using the degradation of MazF:</b> For that purpose the insertion of a plasmid containing the ssrA-tagged toxin encoding gene is needed. Since the predominance of the toxin activates a cell death pathway in bacteria, a bacterium containing a module that allows light inducible degradation of the MazF would only be viable, when it is degraded. In darkness the MazF overexpression is no longer compensated and aggregation of it leads to cell death. </li><li><b>Using the degradation of the antitoxin:</b> Two plasmids are needed: The first one to express the MazF and the second one to express the ssra-tagged MazE, in such manner that the amounts of the <i>MazF</i> and the <i>MazE</i> are in equilibrium. Once light induces the degradation system, the MazE is degraded and the predominant toxin will kill the bacterium.</li></ul>As we explained in the general kill-switch system text we finally focused on the second system (via the degradation of MazE).</br>With the design of a MazEF kill-switch system the possibility of resuscitating bacteria in the way Amitai et al. showed has to be considered. A predominant MazF could kill a bacterium in LB within about two hours, but it needs to be predominant over a long period (>7h) to induce its death without it being resuscitated by renewed <i>mazE</i> expression (Fig.1A)with more than 50% probability .</br>Certainly these facts seem to be unfavourable for the realization of a kill switch system via MazEF, but fortunately our system of heterodimerization (Lungu et al.) allows long continuous degradation^{<a href = #673>67.3</a>}, due to the high stability of the light induced hererodimer. Therefore likely a short exposure time will result in prolonged protein degradation sufficing for bacterial death. Additionally, Amitai et al. showed that the less nutrition is available for a bacterium, the earlier the point of no return is reached. If a bacterium escapes the lab, it will likely have less nutrition available than in LB medium. It might reach the point of no return earlier.</br>We described a light inducible MazEF kill-switch system via the insertion of plasmids into bacteria. However a final kill-switch system would have to be implemented in the genomic DNA since plasmids in bacteria can be ejected, for instance via cell division, whereas a genomic DNA mutation is less likely to occur. Nevertheless risk of a loss of function cannot be eliminated , which is why a secure system should countain much more than one kill-switch system to compensate the malfunction of a single kill-switch system. Therefore, we consider the MazEF kill-switch system to be part of a much larger security system for genetically engineered bacteria.<h2>References</h2><a name =671>67.1</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC532418/'>MazF-Mediated Cell Death in Escherichia coli: a Point of No Return, Shahar Amitai et al., Journal of Bacteriology Vol. 186, No. 24, 2004, p.8295–8300.</a></br><a name =672>67.2</a> <a href = 'http://www.ncbi.nlm.nih.gov/pubmed/?term=Rapid+induction+and+reversal+of+bacteriostatic+conditions+by+controlled+expression+of+toxins+and+antitoxins'>Rapid induction and reversal of bacteriostatic conditions by controlled expression of toxins and antitoxins, Pedersen et al., Molecular Microbiology 45, 2002, 501–510.</a></br><a name =673>67.3</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334866/'>Designing Photoswitchable Peptides Using the AsLOV2 Domain, Oana I. Lungu et al., Chem Biol. 2012, 19(4):507-17.</a></br><a name =674>67.4</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC39188/'>An Escherichia coli chromosomal addiction module regulated by guanosine 3,5-bispyrophosphate: a model for programmed bacterial cell death, Aizenman, E., H. Engelberg-Kulka, and G. Glaser, Proc. Natl. Acad. Sci., 1996, USA 93:6059-6063.</a></br><a name =675>67.5</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC28068/'>rexB of bacteriophage lambda is an anti-cell death gene. Engelberg-Kulka, H., M. Reches, S. Narasimhan, R. Schoulaker-Schwarz, Y. Klemes, E. Aizenman, and G. Glaser, Proc. Natl. Acad. Sci., 1998, USA 95:15481-15486.</a></br><a name =676>67.6</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC95100/'>Programmed cell death in Escherichia coli: some antibiotics can trigger mazEF lethality, Sat, B., R. Hazan, T. Fisher, H. Khaner, G. Glaser, and H. Engelberg-Kulka, J. Bacteriol. 2001, 183:2041-2045.</a></br><a name =677>67.7</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC95101/'>Postsegregational killing mediated by the P1 phage addiction module Phd-Doc requires the Escherichia coli programmed cell death system mazEF, Hazan, R., B. Sat, M. Reches, and H. Engelberg-Kulka, J. Bacteriol. 2001, 183:2046–2050.</a></br><a name =678>67.8</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC150121/'>The Escherichia coli mazEF suicide module mediates thymineless death, Sat, B., M. Reches, and H. Engelberg-Kulka, J. Bacteriol. 2003, 185:1803–1807.</a></br><a name =679>67.9</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC415763/'>Escherichia coli mazEFmediated cell death is triggered by various stressful conditions, Hazan, R., B. Sat, and H. Engelberg-Kulka, J. Bacteriol. 2004, 186:3663–3669.</a>";

content.parents=[54];

content.parents=[54];

content.childs=[];

content.childs=[];

content.titleLong = "The ccd toxin-antitoxin system";

content.titleLong = "The ccd toxin-antitoxin system";

content.text= "The ccd module is a toxin-antitoxin (TA) system similar to the mazE/mazF system. The module is located on the F Plasmid in Escherichia coli bacteria and essential for their survival. Normally the toxin ccdB is inactivated by the presence of the antitoxin ccdA in the form of a ccdAB complex. If ccdA is no longer available, ccdB inhibits DNA gyrase which leads to cell death. Gyrase is a type IIA topoisomerase and is able to produce negative DNA supercoiling by making a double-strand break in the DNA and religating it. The gyrase enzyme consists of two subunits: the C-terminal GyrA domain that wraps around the DNA strand and the N-terminal GyrB domain that catalyses the ATP-dependant supercoiling of the DNA. CcdB stabilizes the gyrase cleavage complex by binding to the GyrA domain and thus blocks the catalytic function of the gyrase. That means that the gyrase remains bound to the DNA and the cleaved DNA is not religated. DNA- and RNA polymerases can’t copy the DNA anymore and cell proliferation as well as protein biosynthesis is stopped. The double-stranded breaks in the DNA initiate cell death.<p>Because gyrases are specific to bacteria such as E. coli it is also a target for some anti-bacterial medications e.g. ciprofloxacin (CFX). As can be seen in the data below, CcdB proves to be as effective as CFX at inducing DNA cleavage ^{<a href=#681>68.1</a>}^{<a href=#682>68.2</a>}</p><div class='content-image'align='center'><img src='https://static.igem.org/mediawiki/2013/2/2f/Sspb_CFX_compared.jpg' width='300'>Comparison of the effect of CcdB and CFX on gyrase activity. N: negatively supercoiled DNA, L: linear DNA, SC: supercoiled DNA. A higher concentration of CcdB/CFX leads to more cleaved (linear) DNA^{<a href= http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635281/figure/fig2/>[source]</a>}</div><div class='content-image' align='center'><img src='https://static.igem.org/mediawiki/2013/e/e4/Bonn_Ccdb_and_ccda.jpg' width='300'>  A higher concentration of ccdB leads to blocking of gyrase and positively supercoiled DNA is cleaved to linear DNA instead of being processed to negatively supercoiled DNA. ^{<a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460896/figure/pone-0046499-g003/>[source]</a>}</div><p>We used this system to build a light-induced kill-switch. Therefore we added the ssrA tag to the antitoxin ccdA. When the bacteria a emitted to light, ccdA is degraded and ccdB is set free and can bind to the gyrase. and cell death is initiated.  Like in most TA systems, the toxin ccdB is relatively stable, while the antitoxin ccdA is vulnerable to degradation.</p><h2>References</h2><p><a name=681>68.1</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635281/'>A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site, Andrew B. Smith and Anthony Maxwell, Nucleic Acids Res. 2006 October; 34(17): 4667–4676, PMC 1635281</a></p><p><a name=682>68.2</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460896/'> A Common Origin for the Bacterial Toxin-Antitoxin Systems parD and ccd, Suggested by Analyses of Toxin/Target and Toxin/Antitoxin Interactions, Andew B. Smith et al, PLoS One. 2012; 7(9): e46499, PMCID: PMC3460896</a></p>";

content.text= "The ccd module is a toxin-antitoxin (TA) system similar to the mazE/mazF system. The module is located on the F Plasmid in Escherichia coli bacteria and essential for their survival. Normally the toxin ccdB is inactivated by the presence of the antitoxin ccdA in the form of a ccdAB complex. If ccdA is no longer available, ccdB inhibits DNA gyrase which leads to cell death. Gyrase is a type IIA topoisomerase and is able to produce negative DNA supercoiling by making a double-strand break in the DNA and religating it. The gyrase enzyme consists of two subunits: the C-terminal GyrA domain that wraps around the DNA strand and the N-terminal GyrB domain that catalyses the ATP-dependant supercoiling of the DNA. CcdB stabilizes the gyrase cleavage complex by binding to the GyrA domain and thus blocks the catalytic function of the gyrase. That means that the gyrase remains bound to the DNA and the cleaved DNA is not religated. DNA- and RNA polymerases can’t copy the DNA anymore and cell proliferation as well as protein biosynthesis is stopped. The double-stranded breaks in the DNA initiate cell death.<p>Because gyrases are specific to bacteria such as E. coli it is also a target for some anti-bacterial medications e.g. ciprofloxacin (CFX). As can be seen in the data below, CcdB proves to be as effective as CFX at inducing DNA cleavage ^{<a href=#681>68.1</a>}^{<a href=#682>68.2</a>}</p><div class='content-image'align='center'><img src='https://static.igem.org/mediawiki/2013/2/2f/Sspb_CFX_compared.jpg' width='300'>Comparison of the effect of CcdB and CFX on gyrase activity. N: negatively supercoiled DNA, L: linear DNA, SC: supercoiled DNA. A higher concentration of CcdB/CFX leads to more cleaved (linear) DNA^{<a href= http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635281/figure/fig2/>[source]</a>}</div><div class='content-image' align='center'><img src='https://static.igem.org/mediawiki/2013/e/e4/Bonn_Ccdb_and_ccda.jpg' width='300'>  A higher concentration of ccdB leads to blocking of gyrase and positively supercoiled DNA is cleaved to linear DNA instead of being processed to negatively supercoiled DNA. ^{<a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460896/figure/pone-0046499-g003/>[source]</a>}</div><p>We used this system to build a light-induced kill-switch. Therefore we added the ssrA tag to the antitoxin ccdA. When the bacteria a emitted to light, ccdA is degraded and ccdB is set free and can bind to the gyrase. and cell death is initiated.  Like in most TA systems, the toxin ccdB is relatively stable, while the antitoxin ccdA is vulnerable to degradation.</p><h2>References</h2><p><a name=681>68.1</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635281/'>A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site, Andrew B. Smith and Anthony Maxwell, Nucleic Acids Res. 2006 October; 34(17): 4667–4676, PMC 1635281</a></p><p><a name=682>68.2</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460896/'> A Common Origin for the Bacterial Toxin-Antitoxin Systems parD and ccd, Suggested by Analyses of Toxin/Target and Toxin/Antitoxin Interactions, Andew B. Smith et al, PLoS One. 2012; 7(9): e46499, PMCID: PMC3460896</a></p>";

content.type="Project";

content.type="Project";

break;

break;

case 71:

case 71:

content.i = 100;  

content.i = 100;  

content.parents=[37];

content.parents=[37];

content.titleShort = "Human Practice";  

content.titleShort = "Human Practice";  

content.titleLong = "Human Practice in General";  

content.titleLong = "Human Practice in General";  

content.text= "<p>The major goal in Human Practice is to make synthetic biology easily understandable and interesting for everybody. For that purpose our project's design follows the well-known franchise of Star Wars; our project name 'LOV WARS' was born. The constant references to the Star Wars movies facilitate the access to synthetic biology to a person that is not familiar with the subject. With the most famous icon of Star Wars being the laser sword it offers a great opportunity to present our light induction system in an entertaining way. We put great effort into adapting every aspect of our public presence to Star Wars.</p><p>Therefore we introduced a <a onclick=node(110)>flash game</a> and a <a onclick=node(109)>comic series</a> called LOV Wars in which we explain basic concepts of synthetic biology and our project. The comic and the LOV Wars shooter arouse interest in people that would normally never engage with the field. That way we can spread the possibilities and advantages that modern genetically engineered organisms offer and clear possible misunderstandings or prejudices against the subject.</p>For the same purpose we organized multiple events for general public at which we presented the tools used in synthetic biology and our project; over the last six months we <a onclick=node(108)>visited several High Schools</a> in Bonn and nearby and gave presentations. In addition we had an information booth in our city center informing passersby about the risks and benefits of synthetic biology with the aid of several posters and flyers. A big highlight was a <a onclick=node(106)>Science slam</a> we arranged. In front of a big audience six scientists from different fields presented an interesting aspect of their academic field. The Science Slam was a great success in terms of getting people in touch with science and without exception received very good critiques.<br> At every opportunity we handed out questionnaires in which we evaluated the public's opinion about synthetic biology and whether our human practice events were informative and interesting.";  

content.text= "<p>The major goal in Human Practice is to make synthetic biology easily understandable and interesting for everybody. For that purpose our project's design follows the well-known franchise of Star Wars; our project name 'LOV WARS' was born. The constant references to the Star Wars movies facilitate the access to synthetic biology to a person that is not familiar with the subject. With the most famous icon of Star Wars being the laser sword it offers a great opportunity to present our light induction system in an entertaining way. We put great effort into adapting every aspect of our public presence to Star Wars.</p><p>Therefore we introduced a <a onclick=node(110)>flash game</a> and a <a onclick=node(109)>comic series</a> called LOV Wars in which we explain basic concepts of synthetic biology and our project. The comic and the LOV Wars shooter arouse interest in people that would normally never engage with the field. That way we can spread the possibilities and advantages that modern genetically engineered organisms offer and clear possible misunderstandings or prejudices against the subject.</p>For the same purpose we organized multiple events for general public at which we presented the tools used in synthetic biology and our project; over the last six months we <a onclick=node(108)>visited several High Schools</a> in Bonn and nearby and gave presentations. In addition we had an information booth in our city center informing passersby about the risks and benefits of synthetic biology with the aid of several posters and flyers. A big highlight was a <a onclick=node(106)>Science slam</a> we arranged. In front of a big audience six scientists from different fields presented an interesting aspect of their academic field. The Science Slam was a great success in terms of getting people in touch with science and without exception received very good critiques.<br> At every opportunity we handed out questionnaires in which we evaluated the public's opinion about synthetic biology and whether our human practice events were informative and interesting.";  

content.type="Human Practice";

content.type="Human Practice";

content.parents=[100];

content.parents=[100];

content.childs=[102,103];

content.childs=[102,103];

content.titleLong = "Project presentations";

content.titleLong = "Project presentations";

content.summary= "We presented iGEM and our project on two scientific conferences.";

content.summary= "We presented iGEM and our project on two scientific conferences.";

content.parents=[101];

content.parents=[101];

content.childs=[];

content.childs=[];

content.titleLong = "Biocom congress Berlin";

content.titleLong = "Biocom congress Berlin";

content.summary= "our day in Berlin. We met important people and other iGEM teams";  

content.summary= "our day in Berlin. We met important people and other iGEM teams";  

case 107:

case 107:

content.i = 107;

content.i = 107;

content.titleShort = "SynBio Day";   

content.titleShort = "SynBio Day";   

content.titleLong = "SynBio Day";

content.titleLong = "SynBio Day";

content.parents=[100];  

content.parents=[100];  

content.childs=[];  

content.childs=[];  

content.titleLong = "School presentations";  

content.titleLong = "School presentations";  

content.summary= "We gave lectures about synthetic biology and our project in schools in order to reach the younger people. ";  

content.summary= "We gave lectures about synthetic biology and our project in schools in order to reach the younger people. ";  

case 109:

case 109:

content.i = 109;

content.i = 109;

content.titleLong = "About the comic";

content.titleLong = "About the comic";

content.summary= "We introduced a hand drawn comic series called &quot;LOV Wars&quot; about Obi-Wan E. Coli and his adventures in the Galaxy of Petri. Basic concepts of synthetic biology are explained along the way.";  

content.summary= "We introduced a hand drawn comic series called &quot;LOV Wars&quot; about Obi-Wan E. Coli and his adventures in the Galaxy of Petri. Basic concepts of synthetic biology are explained along the way.";  

content.parents=[100];

content.parents=[100];

content.childs=[];

content.childs=[];

content.titleLong = "About the LOV-Wars Shooter";

content.titleLong = "About the LOV-Wars Shooter";

content.summary= "The thoughts and ideas behind our game.";

content.summary= "The thoughts and ideas behind our game.";

case 112:

case 112:

content.i = 112;

content.i = 112;

content.titleShort = "Comic";

content.titleShort = "Comic";

content.titleLong = "Comic – The adventures of Obi Wan E.Coli";

content.titleLong = "Comic – The adventures of Obi Wan E.Coli";

content.summary = "Read about the adventures of Obi Wan E.Coli";

content.summary = "Read about the adventures of Obi Wan E.Coli";

content.type = "Human Practice";

content.type = "Human Practice";

break;

break;

content.i = 130;

content.i = 130;

content.parents=[100];

content.parents=[100];

content.titleShort = "About us";

content.titleShort = "About us";

content.titleLong = "About us";

content.titleLong = "About us";

content.summary= "The Team - Presentation of all team members";

content.summary= "The Team - Presentation of all team members";

content.type="Team";

content.type="Team";

break;

break;

content.titleLong = "Sponsors";  

content.titleLong = "Sponsors";  

content.summary= "Many sponsors made our work possible.";

content.summary= "Many sponsors made our work possible.";

content.type="Team";  

content.type="Team";  

break;

break;

content.titleShort = "Bonn";  

content.titleShort = "Bonn";  

content.titleLong = "Our city Bonn";  

content.titleLong = "Our city Bonn";  

content.text= "Bonn has a long and eventful history. The city was founded by the Romans in the year 12 before Christ and continued to grow ever since. Bonn also was the birthplace of the famous musician and composer Beethoven and even was the Capitol of West Germany for 41 years. Today Bonn has around 300 000 habitants and has become the cultural of the region. It is set on the river Rhine very close to Cologne. Everybody gets smitten with its charm and unique mixture of tradition and modern lifestyle. The city&apos;s historic center and the setting close to the river Rhine turn it into a wonderful place to live and study.<p style='text-align:center;'>the city&apos;s slogan:</br><i>&quot;Stadt. City. Ville. Bonn&quot;</i></p></br><p><a href=https://static.igem.org/mediawiki/2013/0/0b/Bonn_by_night.JPG><div class='content-image'align='center'><img src='https://static.igem.org/mediawiki/2013/0/0b/Bonn_by_night.JPG' width='600'></a>Bonn by night ^{&copy;Jan Hansen}</div></p><p><div class='content-image'align='center'><a href=https://static.igem.org/mediawiki/2013/a/a6/Bonn_poppelsdorfer_schloss.JPG><img src='https://static.igem.org/mediawiki/2013/a/a6/Bonn_poppelsdorfer_schloss.JPG' width='600'></a>The university ^{&copy;Jan Hansen}</div><p><a href=https://static.igem.org/mediawiki/2013/0/0b/Bonn_by_night.JPG><div class='content-image'align='center'><img src='https://static.igem.org/mediawiki/2013/7/75/Bonn_beethoven.JPG' width='600'></a>The famous Beethoven statue in the city center ^{<a href=http://www.bone-diagenesis.org/moziloCMS/gallery.php?gal=Bonn-nbsp~Impressions>[source]</a>}</div></p><h2>References</h2><p><a name='1'>1.</a> <a href='http://www.bonn.de'>http://www.bonn.de</a></p><p><a name='2'>2.</a> <a href='http://de.wikipedia.org/wiki/Bonn'>http://de.wikipedia.org/wiki/Bonn</a></p>";  

content.text= "Bonn has a long and eventful history. The city was founded by the Romans in the year 12 before Christ and continued to grow ever since. Bonn also was the birthplace of the famous musician and composer Beethoven and even was the Capitol of West Germany for 41 years. Today Bonn has around 300 000 habitants and has become the cultural of the region. It is set on the river Rhine very close to Cologne. Everybody gets smitten with its charm and unique mixture of tradition and modern lifestyle. The city&apos;s historic center and the setting close to the river Rhine turn it into a wonderful place to live and study.<p style='text-align:center;'>the city&apos;s slogan:</br><i>&quot;Stadt. City. Ville. Bonn&quot;</i></p></br><p><a href=https://static.igem.org/mediawiki/2013/0/0b/Bonn_by_night.JPG><div class='content-image'align='center'><img src='https://static.igem.org/mediawiki/2013/0/0b/Bonn_by_night.JPG' width='600'></a>Bonn by night ^{&copy;Jan Hansen}</div></p><p><div class='content-image'align='center'><a href=https://static.igem.org/mediawiki/2013/a/a6/Bonn_poppelsdorfer_schloss.JPG><img src='https://static.igem.org/mediawiki/2013/a/a6/Bonn_poppelsdorfer_schloss.JPG' width='600'></a>The university ^{&copy;Jan Hansen}</div><p><a href=https://static.igem.org/mediawiki/2013/0/0b/Bonn_by_night.JPG><div class='content-image'align='center'><img src='https://static.igem.org/mediawiki/2013/7/75/Bonn_beethoven.JPG' width='600'></a>The famous Beethoven statue in the city center ^{<a href=http://www.bone-diagenesis.org/moziloCMS/gallery.php?gal=Bonn-nbsp~Impressions>[source]</a>}</div></p><h2>References</h2><p><a name='1'>1.</a> <a href='http://www.bonn.de'>http://www.bonn.de</a></p><p><a name='2'>2.</a> <a href='http://de.wikipedia.org/wiki/Bonn'>http://de.wikipedia.org/wiki/Bonn</a></p>";  

content.type="Team";  

content.type="Team";  

break; */

break; */

case 112:

case 112:

content.i = 112;

content.i = 112;

content.titleShort = "Comic";

content.titleShort = "Comic";

content.titleLong = "Comic – The adventures of Obi Wan E.Coli";

content.titleLong = "Comic – The adventures of Obi Wan E.Coli";

content.type = "Human Practice";

content.type = "Human Practice";

break;

break;

case 200:

case 200:

     }

     }

     }

     }

</script>

</script>

</html>

</html>