Template:Team:Bonn:NetworkData

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.titleShort = "ccdA/ccdB";  

content.titleShort = "ccdA/ccdB";  

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

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

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

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

content.type="Project";

content.type="Project";

content.titleShort = "Bonn";  

content.titleShort = "Bonn";  

content.titleLong = "Our city Bonn";  

content.titleLong = "Our city Bonn";  

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

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

content.type="Team";  

content.type="Team";