Template:Team:Bonn:NetworkData

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";  

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content.i = 42;

content.i = 42;

content.parents=[74];  

content.parents=[74];  

content.summary= "This article deals with the Structure of sspBα and conformational details of its binding to ssrA and ClpXP during tethering.";

content.summary= "This article deals with the Structure of sspBα 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 [42.1]</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. [42.2]Chien et al. [42.1] 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. [42.1]</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>[42.1] Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918 </br> [42.2] 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"; <!-- References-Ueberschrift muss an Format des Bildes angepasst werden -->

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 [42.1]</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. [42.2]Chien et al. [42.1] 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. [42.1]</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>[42.1] Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918 </br> [42.2] 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"; <!-- References-Ueberschrift muss an Format des Bildes angepasst werden -->

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content.i = 43;

content.i = 43;

content.childs=[];  

content.childs=[];  

content.titleShort = "SspB Split";  

content.titleShort = "SspB Split";  

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=#[43.1]>[43.1]</a>}. Thus ccssrA only binds ccSspB but not E. coli SspB. ^{<a href=#[43.2]>[43.2]</a>} ^{<a href=#[43.3]>[43.3]</a>} ^{<a href=#[43.4]>[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=#[43.1]>[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=#[43.1]>[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=#[43.1]>[43.1]</a>} <h2>References:</h2>  </br> <a name=[43.1]>[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=[43.2]>[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=[43.3]>[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=[43.4]>[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=#[43.1]>[43.1]</a>}. Thus ccssrA only binds ccSspB but not E. coli SspB. ^{<a href=#[43.2]>[43.2]</a>} ^{<a href=#[43.3]>[43.3]</a>} ^{<a href=#[43.4]>[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=#[43.1]>[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=#[43.1]>[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=#[43.1]>[43.1]</a>} <h2>References:</h2>  </br> <a name=[43.1]>[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=[43.2]>[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=[43.3]>[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=[43.4]>[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>";  

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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.";

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content.summary= "The design of our System enables several advantages";

content.summary= "The design of our System enables several advantages";

content.text= "The design of our System enables several advantages";  

content.text= "The design of our System enables several advantages";  

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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";

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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";

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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";

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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= "";  

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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= "";  

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case 51:

case 51:

content.i = 51;

content.i = 51;

content.parents=[45];

content.parents=[45];

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= "";  

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content.i = 50;

content.i = 50;

content.parents=[37];

content.parents=[37];

content.summary= "There are several applications for our systems e.g. a light inducible Kill Switch or investigation of protein function";

content.summary= "There are several applications for our systems e.g. a light inducible Kill Switch or investigation of protein function";

content.text= "";  

content.text= "";  

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case 54:

case 54:

content.i = 54;

content.i = 54;

content.parents=[53];

content.parents=[53];

content.summary= "To understand the role of a specific gene or DNA region is one of the big challenges in lifescience research. Our system, which allows the fast and convenient elimination of defined proteins, is a new improved technique, with many advantages.";

content.summary= "To understand the role of a specific gene or DNA region is one of the big challenges in lifescience research. Our system, which allows the fast and convenient elimination of defined proteins, is a new improved technique, with many advantages.";

content.text= "To understand the role of a specific gene or DNA region is one of the big challenges in lifescience research. Our system, which allows the fast and convenient elimination of defined proteins, is a new improved technique, with many advantages. It allows the control of protein activity with respect to time and space, is fast, robust, can be used for different proteins and changes the protein sequence only little, because the ssrA tag consist only out of 15 amino acids. Recently technologies like gene knockout or knockdown had been developed, which allow to investigate the role of a particular gene or DNA region by comparing the knockout organism to a wildtype with a similar genetic background. </br></br> A knockout means that a particular gene is deleted from the genome of an organism. This organism might be bacteria or yeast, but also eukaryotic cells, plants or even animals. To create a knockout organism recombinant DNA is inserted into a gene (Bartke, 2006^{<a href=#ref55.3>55.3</a>}). When a genes sequence is interrupted, it may still be translated, but the resulting protein will be nonfunctional. Moreover, it is possible to knockout the gene only in defined tissues or at defined time points. This technique is called a conditional knockout. </br></br> On the other hand the knockdown, does not eliminate the specific gene on DNA, but on RNA level. Here interfering RNAs (siRNA) are inserted into the cell, leading to the degradation of the genes mRNA (Pratt and MacRae, 2009^{<a href=#ref55.1>55.1</a>}) and hence no protein can be produced. </br></br> In contrast to the knockout and knockdown, our system allows the expression of the gene and the translation into functional protein. However, irradiation with blue light leads to the fast elimination of the particular protein. Therefore, one of the major advantages of our system is its speed, not only in comparison to knockout and knockdown, but also in comparison to other protein level systems. Comparing our system, to other protein level systems, like the system developed by Davis et al.^{<a href=#ref55.2>55.2</a>} in 2011, which are induced by small molecules our system would still be faster due to the use of light. In small molecule systems it takes some time until the small molecules reach their target in the case of light this happens within milliseconds. Furthermore, with our system it is not necessary to add any kind of activator molecules, which might effect the results, to the cells. This enables researchers to investigate cell activity with and with out the protein in direct comparison, while the only interference is one light puls.</br></br><h2>References</h2></br></br><p><a name=ref55.1>55.1</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709356/>Pratt and MacRae (2009) The RNA-induced silencing complex: a versatile gene-silencing machine.</a></p> </br> <p><a name=ref55.2 >55.2</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220803/>Davis et al. (2011) Small-molecule control of protein degradation using split adaptors.</a></p></br><p><a name=ref55.3 >55.3</a> <a href=http://www.sciencedirect.com/science/article/pii/S0531556506002798>Bartke (2006) New findings in transgenic, gene knockout and mutant mice.</a></p>";  

content.text= "To understand the role of a specific gene or DNA region is one of the big challenges in lifescience research. Our system, which allows the fast and convenient elimination of defined proteins, is a new improved technique, with many advantages. It allows the control of protein activity with respect to time and space, is fast, robust, can be used for different proteins and changes the protein sequence only little, because the ssrA tag consist only out of 15 amino acids. Recently technologies like gene knockout or knockdown had been developed, which allow to investigate the role of a particular gene or DNA region by comparing the knockout organism to a wildtype with a similar genetic background. </br></br> A knockout means that a particular gene is deleted from the genome of an organism. This organism might be bacteria or yeast, but also eukaryotic cells, plants or even animals. To create a knockout organism recombinant DNA is inserted into a gene (Bartke, 2006^{<a href=#ref55.3>55.3</a>}). When a genes sequence is interrupted, it may still be translated, but the resulting protein will be nonfunctional. Moreover, it is possible to knockout the gene only in defined tissues or at defined time points. This technique is called a conditional knockout. </br></br> On the other hand the knockdown, does not eliminate the specific gene on DNA, but on RNA level. Here interfering RNAs (siRNA) are inserted into the cell, leading to the degradation of the genes mRNA (Pratt and MacRae, 2009^{<a href=#ref55.1>55.1</a>}) and hence no protein can be produced. </br></br> In contrast to the knockout and knockdown, our system allows the expression of the gene and the translation into functional protein. However, irradiation with blue light leads to the fast elimination of the particular protein. Therefore, one of the major advantages of our system is its speed, not only in comparison to knockout and knockdown, but also in comparison to other protein level systems. Comparing our system, to other protein level systems, like the system developed by Davis et al.^{<a href=#ref55.2>55.2</a>} in 2011, which are induced by small molecules our system would still be faster due to the use of light. In small molecule systems it takes some time until the small molecules reach their target in the case of light this happens within milliseconds. Furthermore, with our system it is not necessary to add any kind of activator molecules, which might effect the results, to the cells. This enables researchers to investigate cell activity with and with out the protein in direct comparison, while the only interference is one light puls.</br></br><h2>References</h2></br></br><p><a name=ref55.1>55.1</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709356/>Pratt and MacRae (2009) The RNA-induced silencing complex: a versatile gene-silencing machine.</a></p> </br> <p><a name=ref55.2 >55.2</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220803/>Davis et al. (2011) Small-molecule control of protein degradation using split adaptors.</a></p></br><p><a name=ref55.3 >55.3</a> <a href=http://www.sciencedirect.com/science/article/pii/S0531556506002798>Bartke (2006) New findings in transgenic, gene knockout and mutant mice.</a></p>";  

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