Template:Team:Bonn:NetworkData

content.i = 2;

content.i = 2;

content.parents=1;  

content.parents=1;  

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.summary= "Proteins can be activated through cleavage of the precursor protein.";  

content.summary= "Proteins can be activated through cleavage of the precursor protein.";  

content.text= "One approach to regulate protein activity is to use posttranslational modification, especially by splitting off an inactivating part from a translated pro-peptide. A change in conformation leads to the functional protein. The cleaving can be effected by enzymatic proteolysis, autoproteolysis or surrounding conditions (e.g. pH).<sup><a href='#1.1'>1.1</a></br> A simple example is the activation of pepsinogen which is caused by a low pH (fig. 1). </br> It is a powerful system to activate proteins, which is frequently used in nature and science. Unfortunately posttranslational cleaving can usually not be reversed. Once activated the inactive pro-form of the Protein cannot be restored.<h2>References</h2><a name='4.1'>1.1</a> <a href='http://www.ncbi.nlm.nih.gov/books/NBK22589/'> Berg JM, Tymoczko JL, Stryer L.: &quot;Biochemistry. 5th Edition&quot;. New York: W H Freeman 2002.</a>";  

content.text= "One approach to regulate protein activity is to use posttranslational modification, especially by splitting off an inactivating part from a translated pro-peptide. A change in conformation leads to the functional protein. The cleaving can be effected by enzymatic proteolysis, autoproteolysis or surrounding conditions (e.g. pH).<sup><a href='#1.1'>1.1</a></br> A simple example is the activation of pepsinogen which is caused by a low pH (fig. 1). </br> It is a powerful system to activate proteins, which is frequently used in nature and science. Unfortunately posttranslational cleaving can usually not be reversed. Once activated the inactive pro-form of the Protein cannot be restored.<h2>References</h2><a name='4.1'>1.1</a> <a href='http://www.ncbi.nlm.nih.gov/books/NBK22589/'> Berg JM, Tymoczko JL, Stryer L.: &quot;Biochemistry. 5th Edition&quot;. New York: W H Freeman 2002.</a>";  

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.titleShort = "Transcriptional regulation";

content.titleShort = "Transcriptional regulation";

content.titleLong = "Transcriptional regulation";  

content.titleLong = "Transcriptional regulation";  

content.type="Background";

content.type="Background";

break;

break;

content.titleShort = "Direct inhibition and activation";

content.titleShort = "Direct inhibition and activation";

content.titleLong = "Direct inhibition and activation";

content.titleLong = "Direct inhibition and activation";

content.type="Background";

content.type="Background";

break;

break;

case 12:

case 12:

content.i = 12;

content.i = 12;

content.type="Background";

content.type="Background";

break;

break;

content.parents=[12];

content.parents=[12];

content.titleShort = "ClpXP protease";

content.titleShort = "ClpXP protease";

content.summary= "The ClpXP protein complex is an AAA+ protease, which means that it uses the energy of ATP hydrolysis to unfold and degenerate marked proteins.";

content.summary= "The ClpXP protein complex is an AAA+ protease, which means that it uses the energy of ATP hydrolysis to unfold and degenerate marked proteins.";

content.type="Background";  

content.type="Background";  

break;

break;

content.i = 14;

content.i = 14;

content.parents=[12];

content.parents=[12];

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";

break;

break;

content.i = 15;  

content.i = 15;  

content.parents=[12];  

content.parents=[12];  

content.summary= "The ssrA tag is a sequence, which allows proteolytic enzymes to degrade them. It relates to proteases like the ClpXP complex in E.coli and it also allows adaptor proteins such as sspB binding and delivering substrates to the proteases in order to make the process more efficient";

content.summary= "The ssrA tag is a sequence, which allows proteolytic enzymes to degrade them. It relates to proteases like the ClpXP complex in E.coli and it also allows adaptor proteins such as sspB binding and delivering substrates to the proteases in order to make the process more efficient";

content.type="Background";  

content.type="Background";  

break;

break;

content.parents=[12];

content.parents=[12];

content.childs=[];

content.childs=[];

content.titleLong = "Control of Protein Degradation Using Split Adaptors";  

content.titleLong = "Control of Protein Degradation Using Split Adaptors";  

content.summary= "Using the protein degradation whith help of SspB, we control the function of SspB by splitting it into two parts, each of which cannot induce degradation on its own.";  

content.summary= "Using the protein degradation whith help of SspB, we control the function of SspB by splitting it into two parts, each of which cannot induce degradation on its own.";  

content.type="Background";

content.type="Background";

break;

break;

content.i = 18;

content.i = 18;

content.parents=[17];

content.parents=[17];

content.titleShort = "Light";

content.titleShort = "Light";

content.titleLong = "Light as a means of induction";

content.titleLong = "Light as a means of induction";

break;

break;

case 19:

case 19:

content.type="Background";

content.type="Background";

break;

break;

case 20:

case 20:

content.childs=[];

content.childs=[];

content.titleShort = "Other systems";

content.titleShort = "Other systems";

content.summary= "More systems";

content.summary= "More systems";

content.text= "More systems";  

content.text= "More systems";  

content.type="Background";

content.type="Background";

break;

break;

case 21:

case 21:

content.parents=[17];

content.parents=[17];

content.childs=[];

content.childs=[];

content.summary= "A review on chemical induction and its advantages and disadvantages";  

content.summary= "A review on chemical induction and its advantages and disadvantages";  

content.text= "Chemical induction can be used to provide both expressional and structural changes in proteins.^{<a href=#321>32.1</a>}^{<a href=#322>32.2</a>} As an advantage it is highly reliable and tunable which renders it very useful for ensuring constant expression levels.^{<a href=#321>32.1</a>} Several promoters such as pBad which is inducible with arabinose or pLac which is inducible with IPTG are frequently used for such purpose.^{<a href=#326>32.6</a>} Yet changes in protein expression require large timescales i.e. tens of minutes to hours, whereas structural changes such as dimerization (for example rapamycin induced dimerization of FRB and FKBP12^{<a href=#324>32.4</a>}) occur much faster i.e. seconds to minutes.^{<a href=#322>32.2</a>} <div class='content-image' align='center'><a href='https://static.igem.org/mediawiki/2013/8/8c/BonnRapamycin3D.jpg'><img src='https://static.igem.org/mediawiki/2013/8/8c/BonnRapamycin3D.jpg' height=260 width=260></a></br><i>A 3D Structure of a Rapamycin induced FKBP-FRB heteromer^{<a href='#327'>32.7</a>}</i></div> However compared to other methods of induction such temporal resolution is inferior. Additionally there are several problems arising from the use of chemical agents. Firstly to come into effect any molecule has to penetrate the cell membrane thus either being actively ingested by the cell or diffusing passively through it, which becomes a severe hindrance when none of these requirements are met.^{<a href=#324>32.4</a>}  Secondly any chemical can be bioactive and hence interfere with the cells metabolism or other substances.^{<a href=#321>32.1</a>}  Also specificity can be a problem especially in vivo, where often several cell types in multicellular organisms are effected. ^{<a href=#325>32.5</a>} Sub cellular spatial resolution can be difficult to achieve since molecules are subject to diffusion. It can be concluded that spatiotemporal resolution is low in chemically induced systems. <div class='content-image' align='center'><a href=https://static.igem.org/mediawiki/2013/0/0f/BonnLacOperon.jpg><img src='https://static.igem.org/mediawiki/2013/0/0f/BonnLacOperon.jpg' height=260 width=260></a></br><i>The Lac Operon: Origin of the Lac1 Promoter</i></div> <h2>References:</h2> <p><a name=321>32.1</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC16554/'>J. Keith Joung, Elizabeth I. Ramm, and Carl O. Pabo: <i>A bacterial two-hybrid selection system for studying protein–DNA and protein–protein interactions.</i> &quot;PNAS&quot; (June 2000)</a></p> <p><a name=322>32.2</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368803/'>Takafumi Miyamoto, Robert DeRose, Allison Suarez, Tasuku Ueno, Melinda Chen, Tai-ping Sun, Michael J. Wolfgang, Chandrani Mukherjee, David J. Meyers, and Takanari Inoue: <i>Rapid and Orthogonal Logic Gating with a Gibberellin-induced Dimerization System. </i>&quot;Nature chemical biology&quot; 8, 465–470 (2012) </a></p> <p><a name=323>32.3</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724991/'>Adilson José da Silva, Antonio Carlos Luperni Horta, Ana Maria Velez, Mônica Rosas C Iemma, Cíntia Regina Sargo, Raquel LC Giordano, Maria Teresa M Novo, Roberto C Giordano, and Teresa Cristina Zangirolami: <i>Non-conventional induction strategies for production of subunit swine erysipelas vaccine antigen in rE. coli fed-batch cultures</i> &quot;Springerplus&quot;2, 322 (2013)</a></p> <p><a name=324>32.4</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3133816/'>Andrei V. Karginov, Yan Zou, David Shirvanyants, Pradeep Kota, Nikolay V. Dokholyan, Douglas D. Young, Klaus M. Hahn, and Alexander Deiters: <i>Light-regulation of protein dimerization and kinase activity in living cells using photocaged rapamycin and engineered FKBP </i>&quot;Journal of the American Chemical Society&quot; 133(3) 420-423 (2011)</a></p> <p><a name=325>32.5</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3529099/'>Yuan Mei and Feng Zhang:<i>Molecular Tools and Approaches for Optogenetics</i> &quot;Biological Psychatry&quot;(2012)</a></p> <p><a name=326>32.6</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711423/'>Jarno Mäkelä, Meenakshisundaram Kandhavelu, Samuel M. D. Oliveira, Jerome G. Chandraseelan, Jason Lloyd-Price, Juha Peltonen, Olli Yli-Harja and Andre S. Ribeiro:<i> In vivo single-molecule kinetics of activation and subsequent activity of the arabinose promoter</i> &quot;Nucleic Acids Research&quot; (2013) </a></p><p><a name='327'>32.7</a><a href='http://www.ncbi.nlm.nih.gov/pubmed/10089303'>Liang J, Choi J, Clardy J.:Refined structure of the FKBP12-rapamycin-FRB ternary complex at 2.2 A resolution.&quot;Acta crystallographica&quot;(1999)</a></p>";

content.text= "Chemical induction can be used to provide both expressional and structural changes in proteins.^{<a href=#321>32.1</a>}^{<a href=#322>32.2</a>} As an advantage it is highly reliable and tunable which renders it very useful for ensuring constant expression levels.^{<a href=#321>32.1</a>} Several promoters such as pBad which is inducible with arabinose or pLac which is inducible with IPTG are frequently used for such purpose.^{<a href=#326>32.6</a>} Yet changes in protein expression require large timescales i.e. tens of minutes to hours, whereas structural changes such as dimerization (for example rapamycin induced dimerization of FRB and FKBP12^{<a href=#324>32.4</a>}) occur much faster i.e. seconds to minutes.^{<a href=#322>32.2</a>} <div class='content-image' align='center'><a href='https://static.igem.org/mediawiki/2013/8/8c/BonnRapamycin3D.jpg'><img src='https://static.igem.org/mediawiki/2013/8/8c/BonnRapamycin3D.jpg' height=260 width=260></a></br><i>A 3D Structure of a Rapamycin induced FKBP-FRB heteromer^{<a href='#327'>32.7</a>}</i></div> However compared to other methods of induction such temporal resolution is inferior. Additionally there are several problems arising from the use of chemical agents. Firstly to come into effect any molecule has to penetrate the cell membrane thus either being actively ingested by the cell or diffusing passively through it, which becomes a severe hindrance when none of these requirements are met.^{<a href=#324>32.4</a>}  Secondly any chemical can be bioactive and hence interfere with the cells metabolism or other substances.^{<a href=#321>32.1</a>}  Also specificity can be a problem especially in vivo, where often several cell types in multicellular organisms are effected. ^{<a href=#325>32.5</a>} Sub cellular spatial resolution can be difficult to achieve since molecules are subject to diffusion. It can be concluded that spatiotemporal resolution is low in chemically induced systems. <div class='content-image' align='center'><a href=https://static.igem.org/mediawiki/2013/0/0f/BonnLacOperon.jpg><img src='https://static.igem.org/mediawiki/2013/0/0f/BonnLacOperon.jpg' height=260 width=260></a></br><i>The Lac Operon: Origin of the Lac1 Promoter</i></div> <h2>References:</h2> <p><a name=321>32.1</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC16554/'>J. Keith Joung, Elizabeth I. Ramm, and Carl O. Pabo: <i>A bacterial two-hybrid selection system for studying protein–DNA and protein–protein interactions.</i> &quot;PNAS&quot; (June 2000)</a></p> <p><a name=322>32.2</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368803/'>Takafumi Miyamoto, Robert DeRose, Allison Suarez, Tasuku Ueno, Melinda Chen, Tai-ping Sun, Michael J. Wolfgang, Chandrani Mukherjee, David J. Meyers, and Takanari Inoue: <i>Rapid and Orthogonal Logic Gating with a Gibberellin-induced Dimerization System. </i>&quot;Nature chemical biology&quot; 8, 465–470 (2012) </a></p> <p><a name=323>32.3</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724991/'>Adilson José da Silva, Antonio Carlos Luperni Horta, Ana Maria Velez, Mônica Rosas C Iemma, Cíntia Regina Sargo, Raquel LC Giordano, Maria Teresa M Novo, Roberto C Giordano, and Teresa Cristina Zangirolami: <i>Non-conventional induction strategies for production of subunit swine erysipelas vaccine antigen in rE. coli fed-batch cultures</i> &quot;Springerplus&quot;2, 322 (2013)</a></p> <p><a name=324>32.4</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3133816/'>Andrei V. Karginov, Yan Zou, David Shirvanyants, Pradeep Kota, Nikolay V. Dokholyan, Douglas D. Young, Klaus M. Hahn, and Alexander Deiters: <i>Light-regulation of protein dimerization and kinase activity in living cells using photocaged rapamycin and engineered FKBP </i>&quot;Journal of the American Chemical Society&quot; 133(3) 420-423 (2011)</a></p> <p><a name=325>32.5</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3529099/'>Yuan Mei and Feng Zhang:<i>Molecular Tools and Approaches for Optogenetics</i> &quot;Biological Psychatry&quot;(2012)</a></p> <p><a name=326>32.6</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711423/'>Jarno Mäkelä, Meenakshisundaram Kandhavelu, Samuel M. D. Oliveira, Jerome G. Chandraseelan, Jason Lloyd-Price, Juha Peltonen, Olli Yli-Harja and Andre S. Ribeiro:<i> In vivo single-molecule kinetics of activation and subsequent activity of the arabinose promoter</i> &quot;Nucleic Acids Research&quot; (2013) </a></p><p><a name='327'>32.7</a><a href='http://www.ncbi.nlm.nih.gov/pubmed/10089303'>Liang J, Choi J, Clardy J.:Refined structure of the FKBP12-rapamycin-FRB ternary complex at 2.2 A resolution.&quot;Acta crystallographica&quot;(1999)</a></p>";

content.parents=[17];

content.parents=[17];

content.childs=[];

content.childs=[];

content.titleLong = "Electrical Induction";

content.titleLong = "Electrical Induction";

content.summary= "A review on chemical induction and its advantages and disadvantages";  

content.summary= "A review on chemical induction and its advantages and disadvantages";  

content.i = 36;

content.i = 36;

content.childs=[37];

content.childs=[37];

content.titleShort = "LOV-ipaA & VinD1";

content.titleShort = "LOV-ipaA & VinD1";

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>";

break;

break;

content.i = 37;

content.i = 37;

content.parents=[36,21,12];

content.parents=[36,21,12];

content.summary= "We engineer a photoswitachble protein degradation system relying on the ClpXP protease system an a LOV domain from avena sativa";  

content.summary= "We engineer a photoswitachble protein degradation system relying on the ClpXP protease system an a LOV domain from avena sativa";  

break;

break;

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];  

content.parents=[74];  

content.childs=[];  

content.childs=[];  

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.parents=[45];

content.parents=[45];

content.childs=[];

content.childs=[];

content.summary= "Due to the use of the protease system protein regulation is fast";

content.summary= "Due to the use of the protease system protein regulation is fast";

content.text= "";  

content.text= "";  

content.parents=[45];

content.parents=[45];

content.childs=[];

content.childs=[];

content.summary= "Since the system is commonly used and reliable its easily applicable to a wide variety of proteins";

content.summary= "Since the system is commonly used and reliable its easily applicable to a wide variety of proteins";

content.text= "";  

content.text= "";  

content.parents=[45];

content.parents=[45];

content.childs=[];

content.childs=[];

content.summary= "Fusing only a 15Aa small tag to the desired protein leads to low influence on its native function";

content.summary= "Fusing only a 15Aa small tag to the desired protein leads to low influence on its native function";

content.text= "";  

content.text= "";  

content.parents=[45];

content.parents=[45];

content.childs=[];

content.childs=[];

content.summary= "Using light easily makes a temporal specific control of activation possible";

content.summary= "Using light easily makes a temporal specific control of activation possible";

content.text= "";  

content.text= "";  

content.parents=[45];

content.parents=[45];

content.childs=[];

content.childs=[];

content.summary= "High spatial control can be achieved easily by local irradation with light";

content.summary= "High spatial control can be achieved easily by local irradation with light";

content.text= "";  

content.text= "";  

content.parents=[45];

content.parents=[45];

content.childs=[];

content.childs=[];

content.summary= "Degradation of the protein leads to a great difference of activity";

content.summary= "Degradation of the protein leads to a great difference of activity";

content.text= "";  

content.text= "";  

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.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.type="Project";

content.type="Project";

break;

break;

case 68:

case 68:

content.parents=[54];

content.parents=[54];

content.childs=[];

content.childs=[];

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.titleLong = "Zinc finger";

content.titleLong = "Zinc finger";

content.summary= "Zinc fingers can be engineered to bind desired DNA sequences";

content.summary= "Zinc fingers can be engineered to bind desired DNA sequences";

content.type="Background";

content.type="Background";

break;

break;

content.childs=[];

content.childs=[];

content.titleShort = "TALE";

content.titleShort = "TALE";

content.summary= "TALEs enable an easy and modular assembly of proteins binding specific desired DNA sequences";

content.summary= "TALEs enable an easy and modular assembly of proteins binding specific desired DNA sequences";

break;

break;

content.parents=[40];  

content.parents=[40];  

content.childs=[42,44];  

content.childs=[42,44];  

content.summary= "This article gives a brief overview of the roles of ssrA and sspB&alpha; for specific function of the ClpXP protease system in C. crescentus.";  

content.summary= "This article gives a brief overview of the roles of ssrA and sspB&alpha; for specific function of the ClpXP protease system in C. crescentus.";  

content.type="Project";

content.type="Project";

break;

break;

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.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";  

content.type="Human Practice";

content.type="Human Practice";

break;

break;

content.titleLong = "The Science Slam - Science for Everybody";

content.titleLong = "The Science Slam - Science for Everybody";

content.summary = "We organized a science slam to offer an experience of science for everyone. And the event met with great approval!";

content.summary = "We organized a science slam to offer an experience of science for everyone. And the event met with great approval!";

content.type = "Human Practice";

content.type = "Human Practice";

break;

break;

case 107:

case 107:

content.i = 107;

content.i = 107;

content.summary= "In cooperation with the iGEM teams of Germany also the team of Bonn organized a day of action for synthetic biology. ";  

content.summary= "In cooperation with the iGEM teams of Germany also the team of Bonn organized a day of action for synthetic biology. ";  

content.text= "<div align='right'><img src='https://static.igem.org/mediawiki/2013/b/b8/BonnAktionstag.JPG' height='260' width='350'></div>In cooperation with the iGEM teams of Germany also the team of Bonn organized a day of action for synthetic biology. </br> At the 7th of September ten of our members met in Bonn downtown to inform the interested civilians of our city about the international genetically engineered machine competition as well as synthetic biology in general and particularly about our project of light inducible degradation of proteins. </br> Therefore we prepared an information booth near the market place, distributed informative leaflets, visualized our ideas in terms of several posters and on top created a survey to examine the people's opinion. </br> <div align='left'><img src='https://static.igem.org/mediawiki/2013/thumb/9/97/BonnAktionstag2.jpg/800px-BonnAktionstag2.jpg' height='260' width='350'></div> At 9 o'clock in the morning we started in front of the LIMES-Institute to arrange the installation of the stand. By car all the needed equipment was transferred to the city center and there assembled under the eyes of the curious townspeople. Two hours later everything was settled and the official part of the day could begin: From 11 until 3 o'clock intrigued city dweller in every range of age stopped by to examine our exhibition walls and to ask questions, which we answered with pleasure. In the end we were surprised about the brisk participation and the lively discussions that aroused, which reflects in the results of the survey, so that we bundled up and left satisfied. Here you can see the summary of our questionaire:<br/> </br> 1. Do you know what synthetic biology means? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/b/bd/BonnQuestion_1.png' height='260' width='350'> <br/> </br> </br> 2. Do you know what the iGEM competition is about? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/4/44/BonnQuestion_2.png' height='260' width='350'> <br/> </br> 3. How do you rate the ralation between chance and risk of sythetic biology? </br><div align='center'><img src='https://static.igem.org/mediawiki/2013/5/58/BonnQuestion3.1.png' height='260' width='350'> </br>  4. What are the main reasons for you that speak against the use of synthetic bioloy? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/6/62/BonnQuestion_4.png' height='260' width='350'> </br> </br> 5. Do you think it is important to inform the public better about the topic ' snythetic biology'?</br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/a/a6/Question_5.png' height='260' width='350'> </br> </br> All in all we consider the action as a great success as we were able to reduce prejudices and elucidate people about advantages of synthetic biology.";

content.text= "<div align='right'><img src='https://static.igem.org/mediawiki/2013/b/b8/BonnAktionstag.JPG' height='260' width='350'></div>In cooperation with the iGEM teams of Germany also the team of Bonn organized a day of action for synthetic biology. </br> At the 7th of September ten of our members met in Bonn downtown to inform the interested civilians of our city about the international genetically engineered machine competition as well as synthetic biology in general and particularly about our project of light inducible degradation of proteins. </br> Therefore we prepared an information booth near the market place, distributed informative leaflets, visualized our ideas in terms of several posters and on top created a survey to examine the people's opinion. </br> <div align='left'><img src='https://static.igem.org/mediawiki/2013/thumb/9/97/BonnAktionstag2.jpg/800px-BonnAktionstag2.jpg' height='260' width='350'></div> At 9 o'clock in the morning we started in front of the LIMES-Institute to arrange the installation of the stand. By car all the needed equipment was transferred to the city center and there assembled under the eyes of the curious townspeople. Two hours later everything was settled and the official part of the day could begin: From 11 until 3 o'clock intrigued city dweller in every range of age stopped by to examine our exhibition walls and to ask questions, which we answered with pleasure. In the end we were surprised about the brisk participation and the lively discussions that aroused, which reflects in the results of the survey, so that we bundled up and left satisfied. Here you can see the summary of our questionaire:<br/> </br> 1. Do you know what synthetic biology means? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/b/bd/BonnQuestion_1.png' height='260' width='350'> <br/> </br> </br> 2. Do you know what the iGEM competition is about? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/4/44/BonnQuestion_2.png' height='260' width='350'> <br/> </br> 3. How do you rate the ralation between chance and risk of sythetic biology? </br><div align='center'><img src='https://static.igem.org/mediawiki/2013/5/58/BonnQuestion3.1.png' height='260' width='350'> </br>  4. What are the main reasons for you that speak against the use of synthetic bioloy? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/6/62/BonnQuestion_4.png' height='260' width='350'> </br> </br> 5. Do you think it is important to inform the public better about the topic ' snythetic biology'?</br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/a/a6/Question_5.png' height='260' width='350'> </br> </br> All in all we consider the action as a great success as we were able to reduce prejudices and elucidate people about advantages of synthetic biology.";

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";

break;

break;

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;

case 133:

case 133:

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.childs=[];  

content.childs=[];  

content.titleShort = "Bonn";  

content.titleShort = "Bonn";  

content.type="Team";  

content.type="Team";  

break;

break;

case 101:

case 101:

content.childs=[];

content.childs=[];

content.titleShort = "Methods";

content.titleShort = "Methods";

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= "<b><a href='#1'>1. 3A – Assembly</a></b></br></br><b><a href='#2'>2. Agarose preparation</a></b></br></br><b><a href='#3'>3.Agarose gel casting</a></b></br></br><b><a href='#4'>4.loading the agarose gel and starting electrophoresis</a></b></br></br><b><a href='#5'>5. Preparation of Antibiotic stocks</a></b></br></br><b><a href='#6'>6. Colony PCR</a></b></br></br><b><a href='#7'>7. Preparation of Glycerol Stocks (iGEM) </a></b></br></br><b><a href='#8'>8.Plasmid Preparation </a></b></br></br><b><a href='#8a'>8a. Midi-Prep (Promega)</a></b></br></br><b><a href='#8b'>8b. Mini-Prep (Promega)</a></b></br></br><b><a href='#9'>9.Preparation of LB agar plates</a></b></br></br><b><a href='#10'>10. PCR - Clean-Up (Macherey und Nagel)</a></b></br></br><b><a href='#11'>11. Preparation of chemocompetent DH5-alpha cells</a></b></br></br><b><a href='#12'>12. Re-transformation of BioBricks</a></b></br></br><b><a href='#13'>13. Strand directed Mutagenesis PCR</a></b></br></br><b><a href='#14'>14. Transformation using Ligation product (in DH5alpha or XL1Blue) </a></b></br></br></br></br><h2><a name='1'>1. 3A – Assembly</a></h2><hr><i>NOTE: Enzymes and buffers were provided by Promega </i></br></br><b>Restriction (50 &micro;l Reaction)</b></br>- 25 &micro;l Mastermix Restriction-Enzyme Buffer (2x, with BSA)</br>- add 1 &micro;l from every restriction enzyme to 500ng backbone equimolar DNA</br>- fill up to 50 &micro;l dest. water</br>- incubate for 1.5h-3h at 37&ordm;C </br>- inactivate for 20min at 70&ordm;C (no clean-up, if directly used for ligation) </br></br><b>Ligation (20 &micro;l Reaction)</b></br>- 2,0 &micro;l equimolare restriction samples (inserts)</br>- 20ng/1.5 &micro;l backbone</br>- fill up to 17.7  &micro;l with dest. water</br>- incubate 5min at 37&ordm;C</br>- add 2 &micro;l Ligation buffer (10x) and 0.3 &micro;l T4 DNA Ligase </br>- incubate for 3h RT or 15&ordm;C over night </br>- inactivate for 10min at 70&ordm;C</br></br></br><h2><a name='2'>2.Agarose preparation</a></h2><hr><b>Materials:</b></br>- Agarose </br>- 500ml bottle</br>- TBE (1x)</br></br><b>Procedure:</b></br>- dissolve 1g agarose in 100ml TBE (1x), resulting in 1% agarose</br>- heat for 2 minutes in a microwave at maximal power</br>- mix</br>- cook until it boils (1 min)</br>- mix carefully</br><i>2 clues for a successful boiling: 1. no cords, 2. boiling retardation</i></br>- store in 65&ordm;C incubator</br></br></br><h2><a name='3'>3.Agarose gel casting</a></h2><hr>- assemble the gel chamber (chamber + 2 fences + 1 - 2 gel combs ) under the ethidiumbromid hood</br>- prepare 50ml falcon tube (label it!)</br>- fill 40ml warm agarose in the falcon tube</br>- add 4 &micro;l  ethidiumbromid (1:10.000) under the ethidiumbromid hood</br>- mix by inverting 2-3 times</br>- fill agarose in the gel chamber</br>- wait until the gel becomes solid (about 20minutes)</br></br></br><h2><a name='4'>4.loading the agarose gel and starting electrophoresis</a></h2><hr>- add LoadingDye (1:6) to your sample</br>- remove comb and fence from the gel</br>- place agarose gel in the electrophoresis chamber</br>- pipette samples carefully in the pockets (small pockets: up to 20 &micro;l, big pockets: up to 50 &micro;l)</br>- Place lid on the electrophoresis chamber and connect the electrodes to it.</br>- set parameters (high resolution: 120V, 20-30minutes, low resolution: 130V, 15min)</br>- evaluate gel under UV-light</br></br></br><h2><a name='5'>5. Preparation of Antibiotic stocks</a></h2><hr><b>Ampicillin:</b></br>- dissolve 100 mg ampicillin in 1 ml dest. water</br>- store at 20 &ordm;C</br></br><b>Chloramphenicol:</b></br>- dissolve 18 mg chloramphenicol in 1 ml ethanol</br>- store at 20 &ordm;C</br></br></br><h2><a name='6'>6. Colony PCR</a></h2><hr>- inoculate 10 &micro;l dest. water with colony.</br>- use 1  &micro;l of this water for one reaction:</br><img src='https://static.igem.org/mediawiki/2013/9/91/Bonn_MS_Methods1.png' width='550px'></br></br></br><h2><a name='7'>7.Preparation of Glycerol Stocks (iGEM)</a></h2><hr>- autoclave glycerol (60%)</br>- add 0,5 ml Glycerol to 1,5 ml cell culture in a cryo tube</br>- mix</br>- shock freeze in liquid nitrogen</br>- store at -80 &ordm;C</br></br></br><h2><a name='8'>8. Plasmid Preparation</a></h2><hr><h2><a name='8a'>8a. Midi-Prep (Promega)</a></h2><hr>- centrifuge 50 ml of liquid cell culture for 10min at 5000g </br>- decant supernatant</br>- resuspend with 3 ml resuspension solution </br>- add 3 ml cell lysis solution and incubate for maximal 3 min at room temperature </br>- add 5 ml neutralization solution </br>- centrifuge for 20 min at 20 &ordm;C, 5000g </br>- vacuum pump lysat through cleaning column into binding column </br>- abolish cleaning column</br>- vacuum pump with 10 ml endotoxin removal wash solution</br>- vacuum pump with 20 ml column wash solution</br>- dry membrane by vacuum</br>- add 600  &micro;l nuclease free water on membrane</br>- centrifuge for 5 min at 1750 g into a fresh tube</br></br></br><h2><a name='8b'>8b. Mini-Prep (Promega)</a></h2><hr>- fill 1,5 ml overnight-culture in a new tube</br>- centrifuge for 30 seconds at maximal speed </br>- decant supernatant</br>- Repeat previous steps 2-5 times (depending on growth density)</br>- resuspend with 600  &micro;l dest water </br>- add 100  &micro;l cell lysis buffer </br>- after 1min (maximum 2min) add 350  &micro;l of neutralization buffer </br>- centrifuge 3min at maximal speed</br>- place mini column in a collection tube and transfer supernatant into PureYield^TM Mini column</br>- centrifuge for 15 sec at maximal speed</br>- add 200  &micro;l Endotoxin Removal Wash</br>- centrifuge for 15 sec at maximal speed</br>- add 400  &micro;l column Wash solution</br>- centrifuge for 30 sec at maximal speed</br>- place mini column in a new tube</br>- add 30  &micro;l elution buffer to the mini column, incubation at RT for 1 min</br>- centrifuge for 15 seconds at maximal speed </br>- store DNA at -20 &ordm;C</br></br></br><h2><a name='9'>9. Preparation of LB agar plates</a></h2><hr>1l LB agar will result in approximately 30 plates</br></br>- dissolve 15g agar and 20g LB in 1l dest. Water</br>- autoclave</br>- cool down to 60-70&ordm;C </br>- add antibiotics (1:1000) under the laminar airflow cabinet</br>- mix</br>- cast plates (approximately  20ml / plate)</br>- dry for 2h by room temperature</br>- store at 4&ordm;C</br></br></br><h2><a name='10'>10. PCR - Clean-Up (Macherey und Nagel) </a></h2><hr><b>Gel Extraction: </b></br>1. add double amount NTI to gel</br>2. Incubate 3-7minutes at 50&ordm;C and at 1000rpm (until gel is dissolved)</br>- continue with regular Clean-up Protocol (from step 3.) </br></br><b>Cleanup: </b></br>1. fill up sample with dest. water to 50 &micro;l, if necessary</br>2. add double amount NTI to the sample</br>3. place column in a collection and add transfer solution to the column</br>4. centrifuge 30 seconds at 11.000g and discard flow through </br>5. add 700 &micro;l NT3 </br>6. centrifuge 30 seconds at 11.000g and discard flow through </br>7. repeat step 5) and 6)</br>8. centrifuge 1minute at 11.000g</br>9. place column in a new tube and dry column at 70&ordm;C for 5min</br></br>small parts (<1000bp): </br>9a. place column in a new tube and add 30 &micro;l Elution buffer </br>9b. incubate 1min at room temperature</br>9c. Centrifuge 1minute at 11.000g </br></br>Bigger Parts: (>1000bp) </br>9A. place column in a new tube and add 20 &micro;l Elution buffer </br>9B. incubate at 70&ordm;C for 5minutes </br>9C. centrifuge at 50g for 1minute</br>9D. centrifuge at 11.000g for 1minute</br>9E. repeat step 9A. to 9D.</br></br></br><h2><a name='11'>11. Preparation of chemocompetent DH5-alpha cells</a></h2><hr>- Start with 200 ml Overnight culture with OD₆₀₀ of 0,6-0,8</br>- centrifuge at 4 &ordm;C, 4500 g, 10 minutes </br>- decant supernatant</br>- resuspend with 40 ml inoune transformation buffer </br>- centrifuge at 4 &ordm;C, 4500 g, 10 minutes </br>- decant supernatant</br>- resuspend in 20 ml inoune transformation buffer </br>- add 1,5 ml DMSO</br>- incubate 10 minutes on ice</br>- transfer 100 – 200 &micro;l into precooled tubes</br>- shock freeze in liquid nitrogen</br></br></br><h2><a name='12'> 12. Re-transformation of Bio Bricks</a></h2><hr>- add 10  &micro;l sterile dest water to DNA on plate </br>- incubate for 10 min at RT</br>- take 2  &micro;l, leave rest on plate </br>- store plates at -20 &ordm;C </br>- add the 2  &micro;l DNA solution to 5  &micro;l competent DH5-alpha</br>- incubate for 30 min on ice </br>- heat shock for 45 s at 42 &ordm;C </br>- incubate 3 min on ice </br>- add 250  &micro;l LB medium at 37 &ordm;C </br>- incubate for 45 min at 37 &ordm;C, 800 rpm </br>- plate 300 &micro;l on Agar-plate with appropriate antibiotic </br>- dry 15min at RT</br>- incubate at 37&ordm;C over night</br></br></br><h2><a name='13'> 13. Strand directed Mutagenesis PCR</a></h2><hr>Prepare master mix and add template as follows: </br><img src='https://static.igem.org/mediawiki/2013/b/b6/Bonn_MS_Methods2.png' width='550px'></br>- start PCR-Program: </br>1. initial denaturation 94&ordm;C for 120 seconds </br>2. Denaturating 94&ordm;C for 30 seconds</br>3. Annealing 94&ordm;C for 30 seconds</br>4. Elongation 68 for 720 seconds</br>5. Repeat step 2) to 4) 12x </br></br></br><h2><a name='14'>14. Transformation using Ligation product (in DH5alpha or XL1Blue) </a></h2><hr>- thaw bacteria on ice </br>- add 2-4 &micro;l Ligation mixture to 50 &micro;l bacteria</br>- incubate 30minutes on ice </br>- heat shock 30-45 seconds (XL1Blue preferably 35 seconds) at 42&ordm;C</br>- incubate 6min on ice</br>- add 250 &micro;l LB medium at 37&ordm;C</br>- incubate for 45minutes at 37&ordm;C, 800rpm </br>- plate 300 &micro;l on appropriate antibiotic</br>- dry 15 minutes at RT</br>- incubate at 37&ordm;C over night</br>";

content.text= "<b><a href='#1'>1. 3A – Assembly</a></b></br></br><b><a href='#2'>2. Agarose preparation</a></b></br></br><b><a href='#3'>3.Agarose gel casting</a></b></br></br><b><a href='#4'>4.loading the agarose gel and starting electrophoresis</a></b></br></br><b><a href='#5'>5. Preparation of Antibiotic stocks</a></b></br></br><b><a href='#6'>6. Colony PCR</a></b></br></br><b><a href='#7'>7. Preparation of Glycerol Stocks (iGEM) </a></b></br></br><b><a href='#8'>8.Plasmid Preparation </a></b></br></br><b><a href='#8a'>8a. Midi-Prep (Promega)</a></b></br></br><b><a href='#8b'>8b. Mini-Prep (Promega)</a></b></br></br><b><a href='#9'>9.Preparation of LB agar plates</a></b></br></br><b><a href='#10'>10. PCR - Clean-Up (Macherey und Nagel)</a></b></br></br><b><a href='#11'>11. Preparation of chemocompetent DH5-alpha cells</a></b></br></br><b><a href='#12'>12. Re-transformation of BioBricks</a></b></br></br><b><a href='#13'>13. Strand directed Mutagenesis PCR</a></b></br></br><b><a href='#14'>14. Transformation using Ligation product (in DH5alpha or XL1Blue) </a></b></br></br></br></br><h2><a name='1'>1. 3A – Assembly</a></h2><hr><i>NOTE: Enzymes and buffers were provided by Promega </i></br></br><b>Restriction (50 &micro;l Reaction)</b></br>- 25 &micro;l Mastermix Restriction-Enzyme Buffer (2x, with BSA)</br>- add 1 &micro;l from every restriction enzyme to 500ng backbone equimolar DNA</br>- fill up to 50 &micro;l dest. water</br>- incubate for 1.5h-3h at 37&ordm;C </br>- inactivate for 20min at 70&ordm;C (no clean-up, if directly used for ligation) </br></br><b>Ligation (20 &micro;l Reaction)</b></br>- 2,0 &micro;l equimolare restriction samples (inserts)</br>- 20ng/1.5 &micro;l backbone</br>- fill up to 17.7  &micro;l with dest. water</br>- incubate 5min at 37&ordm;C</br>- add 2 &micro;l Ligation buffer (10x) and 0.3 &micro;l T4 DNA Ligase </br>- incubate for 3h RT or 15&ordm;C over night </br>- inactivate for 10min at 70&ordm;C</br></br></br><h2><a name='2'>2.Agarose preparation</a></h2><hr><b>Materials:</b></br>- Agarose </br>- 500ml bottle</br>- TBE (1x)</br></br><b>Procedure:</b></br>- dissolve 1g agarose in 100ml TBE (1x), resulting in 1% agarose</br>- heat for 2 minutes in a microwave at maximal power</br>- mix</br>- cook until it boils (1 min)</br>- mix carefully</br><i>2 clues for a successful boiling: 1. no cords, 2. boiling retardation</i></br>- store in 65&ordm;C incubator</br></br></br><h2><a name='3'>3.Agarose gel casting</a></h2><hr>- assemble the gel chamber (chamber + 2 fences + 1 - 2 gel combs ) under the ethidiumbromid hood</br>- prepare 50ml falcon tube (label it!)</br>- fill 40ml warm agarose in the falcon tube</br>- add 4 &micro;l  ethidiumbromid (1:10.000) under the ethidiumbromid hood</br>- mix by inverting 2-3 times</br>- fill agarose in the gel chamber</br>- wait until the gel becomes solid (about 20minutes)</br></br></br><h2><a name='4'>4.loading the agarose gel and starting electrophoresis</a></h2><hr>- add LoadingDye (1:6) to your sample</br>- remove comb and fence from the gel</br>- place agarose gel in the electrophoresis chamber</br>- pipette samples carefully in the pockets (small pockets: up to 20 &micro;l, big pockets: up to 50 &micro;l)</br>- Place lid on the electrophoresis chamber and connect the electrodes to it.</br>- set parameters (high resolution: 120V, 20-30minutes, low resolution: 130V, 15min)</br>- evaluate gel under UV-light</br></br></br><h2><a name='5'>5. Preparation of Antibiotic stocks</a></h2><hr><b>Ampicillin:</b></br>- dissolve 100 mg ampicillin in 1 ml dest. water</br>- store at 20 &ordm;C</br></br><b>Chloramphenicol:</b></br>- dissolve 18 mg chloramphenicol in 1 ml ethanol</br>- store at 20 &ordm;C</br></br></br><h2><a name='6'>6. Colony PCR</a></h2><hr>- inoculate 10 &micro;l dest. water with colony.</br>- use 1  &micro;l of this water for one reaction:</br><img src='https://static.igem.org/mediawiki/2013/9/91/Bonn_MS_Methods1.png' width='550px'></br></br></br><h2><a name='7'>7.Preparation of Glycerol Stocks (iGEM)</a></h2><hr>- autoclave glycerol (60%)</br>- add 0,5 ml Glycerol to 1,5 ml cell culture in a cryo tube</br>- mix</br>- shock freeze in liquid nitrogen</br>- store at -80 &ordm;C</br></br></br><h2><a name='8'>8. Plasmid Preparation</a></h2><hr><h2><a name='8a'>8a. Midi-Prep (Promega)</a></h2><hr>- centrifuge 50 ml of liquid cell culture for 10min at 5000g </br>- decant supernatant</br>- resuspend with 3 ml resuspension solution </br>- add 3 ml cell lysis solution and incubate for maximal 3 min at room temperature </br>- add 5 ml neutralization solution </br>- centrifuge for 20 min at 20 &ordm;C, 5000g </br>- vacuum pump lysat through cleaning column into binding column </br>- abolish cleaning column</br>- vacuum pump with 10 ml endotoxin removal wash solution</br>- vacuum pump with 20 ml column wash solution</br>- dry membrane by vacuum</br>- add 600  &micro;l nuclease free water on membrane</br>- centrifuge for 5 min at 1750 g into a fresh tube</br></br></br><h2><a name='8b'>8b. Mini-Prep (Promega)</a></h2><hr>- fill 1,5 ml overnight-culture in a new tube</br>- centrifuge for 30 seconds at maximal speed </br>- decant supernatant</br>- Repeat previous steps 2-5 times (depending on growth density)</br>- resuspend with 600  &micro;l dest water </br>- add 100  &micro;l cell lysis buffer </br>- after 1min (maximum 2min) add 350  &micro;l of neutralization buffer </br>- centrifuge 3min at maximal speed</br>- place mini column in a collection tube and transfer supernatant into PureYield^TM Mini column</br>- centrifuge for 15 sec at maximal speed</br>- add 200  &micro;l Endotoxin Removal Wash</br>- centrifuge for 15 sec at maximal speed</br>- add 400  &micro;l column Wash solution</br>- centrifuge for 30 sec at maximal speed</br>- place mini column in a new tube</br>- add 30  &micro;l elution buffer to the mini column, incubation at RT for 1 min</br>- centrifuge for 15 seconds at maximal speed </br>- store DNA at -20 &ordm;C</br></br></br><h2><a name='9'>9. Preparation of LB agar plates</a></h2><hr>1l LB agar will result in approximately 30 plates</br></br>- dissolve 15g agar and 20g LB in 1l dest. Water</br>- autoclave</br>- cool down to 60-70&ordm;C </br>- add antibiotics (1:1000) under the laminar airflow cabinet</br>- mix</br>- cast plates (approximately  20ml / plate)</br>- dry for 2h by room temperature</br>- store at 4&ordm;C</br></br></br><h2><a name='10'>10. PCR - Clean-Up (Macherey und Nagel) </a></h2><hr><b>Gel Extraction: </b></br>1. add double amount NTI to gel</br>2. Incubate 3-7minutes at 50&ordm;C and at 1000rpm (until gel is dissolved)</br>- continue with regular Clean-up Protocol (from step 3.) </br></br><b>Cleanup: </b></br>1. fill up sample with dest. water to 50 &micro;l, if necessary</br>2. add double amount NTI to the sample</br>3. place column in a collection and add transfer solution to the column</br>4. centrifuge 30 seconds at 11.000g and discard flow through </br>5. add 700 &micro;l NT3 </br>6. centrifuge 30 seconds at 11.000g and discard flow through </br>7. repeat step 5) and 6)</br>8. centrifuge 1minute at 11.000g</br>9. place column in a new tube and dry column at 70&ordm;C for 5min</br></br>small parts (<1000bp): </br>9a. place column in a new tube and add 30 &micro;l Elution buffer </br>9b. incubate 1min at room temperature</br>9c. Centrifuge 1minute at 11.000g </br></br>Bigger Parts: (>1000bp) </br>9A. place column in a new tube and add 20 &micro;l Elution buffer </br>9B. incubate at 70&ordm;C for 5minutes </br>9C. centrifuge at 50g for 1minute</br>9D. centrifuge at 11.000g for 1minute</br>9E. repeat step 9A. to 9D.</br></br></br><h2><a name='11'>11. Preparation of chemocompetent DH5-alpha cells</a></h2><hr>- Start with 200 ml Overnight culture with OD₆₀₀ of 0,6-0,8</br>- centrifuge at 4 &ordm;C, 4500 g, 10 minutes </br>- decant supernatant</br>- resuspend with 40 ml inoune transformation buffer </br>- centrifuge at 4 &ordm;C, 4500 g, 10 minutes </br>- decant supernatant</br>- resuspend in 20 ml inoune transformation buffer </br>- add 1,5 ml DMSO</br>- incubate 10 minutes on ice</br>- transfer 100 – 200 &micro;l into precooled tubes</br>- shock freeze in liquid nitrogen</br></br></br><h2><a name='12'> 12. Re-transformation of Bio Bricks</a></h2><hr>- add 10  &micro;l sterile dest water to DNA on plate </br>- incubate for 10 min at RT</br>- take 2  &micro;l, leave rest on plate </br>- store plates at -20 &ordm;C </br>- add the 2  &micro;l DNA solution to 5  &micro;l competent DH5-alpha</br>- incubate for 30 min on ice </br>- heat shock for 45 s at 42 &ordm;C </br>- incubate 3 min on ice </br>- add 250  &micro;l LB medium at 37 &ordm;C </br>- incubate for 45 min at 37 &ordm;C, 800 rpm </br>- plate 300 &micro;l on Agar-plate with appropriate antibiotic </br>- dry 15min at RT</br>- incubate at 37&ordm;C over night</br></br></br><h2><a name='13'> 13. Strand directed Mutagenesis PCR</a></h2><hr>Prepare master mix and add template as follows: </br><img src='https://static.igem.org/mediawiki/2013/b/b6/Bonn_MS_Methods2.png' width='550px'></br>- start PCR-Program: </br>1. initial denaturation 94&ordm;C for 120 seconds </br>2. Denaturating 94&ordm;C for 30 seconds</br>3. Annealing 94&ordm;C for 30 seconds</br>4. Elongation 68 for 720 seconds</br>5. Repeat step 2) to 4) 12x </br></br></br><h2><a name='14'>14. Transformation using Ligation product (in DH5alpha or XL1Blue) </a></h2><hr>- thaw bacteria on ice </br>- add 2-4 &micro;l Ligation mixture to 50 &micro;l bacteria</br>- incubate 30minutes on ice </br>- heat shock 30-45 seconds (XL1Blue preferably 35 seconds) at 42&ordm;C</br>- incubate 6min on ice</br>- add 250 &micro;l LB medium at 37&ordm;C</br>- incubate for 45minutes at 37&ordm;C, 800rpm </br>- plate 300 &micro;l on appropriate antibiotic</br>- dry 15 minutes at RT</br>- incubate at 37&ordm;C over night</br>";

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;

     }

     }

     }

     }

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