Template:Team:Bonn:NetworkData

From 2013.igem.org

(Difference between revisions)
Line 17: Line 17:
case 1:
case 1:
-
 
content.i = 1;
content.i = 1;
Line 31: Line 30:
case 2:
case 2:
-
 
content.i = 2;
content.i = 2;
Line 45: Line 43:
case 3:
case 3:
 +
content.i = 3;  
content.i = 3;  
content.parents=[2];
content.parents=[2];
Line 54: Line 53:
content.type="Background";  
content.type="Background";  
break;
break;
 +
case 4:
case 4:
 +
content.i = 4;
content.i = 4;
content.parents=[3]
content.parents=[3]
Line 65: Line 66:
content.type="Background";
content.type="Background";
break;
break;
 +
case 5:
case 5:
 +
content.i = 5;  
content.i = 5;  
content.parents=[3];
content.parents=[3];
Line 76: Line 79:
content.type="Background";
content.type="Background";
break;
break;
 +
case 7:
case 7:
 +
content.i = 7;
content.i = 7;
content.parents=[2];
content.parents=[2];
Line 88: Line 93:
break;
break;
-
case 8:
 
 +
case 8:
content.i = 8;
content.i = 8;
Line 103: Line 108:
case 9:
case 9:
-
 
content.i = 9;  
content.i = 9;  
Line 117: Line 121:
case 10:
case 10:
 +
content.i = 10;
content.i = 10;
content.parents=[2];
content.parents=[2];
Line 129: Line 134:
case 11:
case 11:
 +
content.i = 11;
content.i = 11;
content.parents=[2];
content.parents=[2];
Line 141: Line 147:
case 12:
case 12:
-
 
content.i = 12;
content.i = 12;
Line 152: Line 157:
content.type="Background";
content.type="Background";
break;
break;
 +
case 13:
case 13:
 +
content.i = 13;
content.i = 13;
content.parents=[12];
content.parents=[12];
Line 161: Line 168:
content.type="Background";  
content.type="Background";  
break;
break;
-
 
-
 
Line 174: Line 179:
content.type="Background";
content.type="Background";
break;
break;
 +
case 15:
case 15:
 +
content.i = 15;  
content.i = 15;  
content.parents=[12];  
content.parents=[12];  
Line 184: Line 191:
break;
break;
 +
 +
case 16:
 +
 +
content.i = 16;
 +
content.parents=[12];
 +
content.childs=[];
 +
content.titleShort = "sspB-Split";
 +
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.text= "The ClpXP protein degradation system can be used for inducible protein degradation as described by Davis et al.  They made use of the native ClpXP system in E. coli with a modified ssrA-tag (DAS+4) at the target protein. <sup><a href=#161>[16.1]</a></sup>  The modified DAS+4 ssrA cannot bind the ClpXP without SspB. <sup><a href=#165>[16.5]</a></sup> Using this dependency of the protein degradation on SspB, they decided to control the function of SspB by splitting it into two parts, each of which cannot induce degradation on its own. <sup><a href=#161>[16.1]</a></sup> </br> </br> Splitting of SspB is possible because its tripartite structure. It consists of (1.) a ssrA-tag binding and dimerization domain (SspB [CORE]), (2.) a flexible linker and (3.) a short peptide module that docks with ClpXP (SspB [XB]). <sup><a href=#163>[16.3]</a></sup> To test whether the linker length can be varied, the degradation rates of GFP-DAS+4 (0.3μM) with SspB (0.15μM) were tested in vitro with 4 different linker lengths (of 5, 25, 48 and 91 amino acids). The results showed that the 25 amino acids variant triggered the fastest degradation, followed by the 25 variant with 60%, 5 variant with 30% and 91 variant with 20% of the 25 variant rate. But even the 91 amino acids variant showed a 40times faster degradation rate than the degradation system without any SspB. <sup><a href=#166>[16.6]</a></sup>  Further experiments (e.g. the split system with FRB-FKBP12 as a linker, see below) demonstrated that even longer linker regions (more than 200 amino acids) are functional). Thus it can be concluded that not only the length of the linker is important but also its structure. <sup><a href=#161>[16.1]</a></sup> </br> </br> <img src='https://static.igem.org/mediawiki/2013/1/1f/Bonn-Gfp.jpg'> <sup><a href=#166>[16.6]</a></sup> </br> </br> To bring both SspB parts together again for inducible degradation, they were combined with a chemical inducible heterodimerisation system: FRB and FKBP12. <sup><a href=#161>[16.1]</a></sup> FKBP12 (FK 06 binding protein, 12 kDa) is a binding protein (108 amino acids <sup><a href=#167>[16.7]</a></sup>) for the small molecule rapamycin. FRB (FKBP-rapamycin binding domain) is the FKBP12-rapamycin binding domain (100 amino acids) of the mammalian protein mTor. In the absence of rapamycin, FKBP12 and FRB show no measurable interaction, while in the presence of rapamycin they build a strong FKBP-rapamycin-FRB ternary complex. <sup><a href=#162>[16.2]</a></sup> </br> </br> In order to achieve inducible degradation Davis et al. created the fusion proteins SspB[CORE]-FRB and FKBP12-SspB[XB]. SspB[CORE]-FRB interacts with the ssrA-tag of the target protein. FKBP12-SspB[XB] interacts with the ClpXP. In absence of rapamycin the two parts of SspB can only bind there particular targets but can’t interact with each other. Therefore, they don’t work as an adapter between the ssrA-taged protein and the ClpXP. By adding rapamycin FRB and FKBP12 dimerize. As a consequence the two parts of SspB get in a spatial closeness and function as an adapter. As a consequent the target protein gets degraded. <sup><a href=#161>[16.1]</a></sup> </br> </br> <img src='https://static.igem.org/mediawiki/2013/2/21/Bonn-rapa-split.jpg'> <sup><a href=#161>[16.1]</a></sup> </br> The efficiency of this system was demonstrated in the following in vitro experiments: GFP-DAS+4 was incubated with ClpXP, FKBP12-SspB[XP], SspB[CORE]-FRB and an ATP-regenerating system. Without rapamycin there was no degradation of GFP. The addition of rapamycin led to a reduction of GFP-ssrA of around 50% in only 360 seconds (degradation rate of 0.58min-1enzyme-1)(figure 2a). <sup><a href=#161>[16.1]</a></sup> Furthermore it was tested how long the degradation system needs to assemble and thus to reach the maximal degradation rate in this system. A time of 20 seconds was measured (figure 2b). <sup><a href=#161>[16.1]</a></sup> </br> </br> <img src='https://static.igem.org/mediawiki/2013/d/d0/Bonn-GFP-Abbau.jpg'> <sup><a href=#161>[16.1]</a></sup> </br> For in vivo testing they introduced the system into an SspB- mutant of E. coli by the plasmid pJD427. This plasmid contains SspB[CORE]-FRB with the weak constitutive promoter proB, FKBP12-SspB[XB] with the strong constitutive promoter proC and a medium-copy p15a origin of replication. For a target protein the lacI transcription repressor was used with a DAS+4-tag recombined to the C-terminus. Usually LacI represses lacZ transcription and thus production of β-galactosidase. Therefore degradation of LacI leads to an increasing β-galactosidase activity. As the assay showed absence of rapamycin results in no change of the β-galactosidase activity. Addition of rapamycin, however, leads to increasing β-galactosidase activity. Hence the system also worked in vivo (in an acceptable time). <sup><a href=#161>[16.1]</a></sup> </br> <img src='https://static.igem.org/mediawiki/2013/2/2d/Bonn-rapa-gel.jpg'> <sup><a href=#161>[16.1]</a></sup> </br> <h2> <b>References:</b> </h2> <a name=161>[16.1]</a> <a href=http://'www.ncbi.nlm.nih.gov/pmc/articles/PMC3220803/'> Small-Molecule Control of Protein Degradation Using Split Adaptors, J. Davis et al, ACS Chem. Biol. 2011, 6, 1205-1213, PMID: 21866931 </a> </br> <a name=162>[16.2]</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/15796538'> Charaterization of the FKBP•Rapamycin•FRB Ternary Complex, L. Banaszynski, C. Liu et al,  J. AM. CHEM. SOC. 2006, 127, 4715-4721, PMID: 15796538 </a> </br> <a name=163>[16.3]</a> <a href=http://'www.ncbi.nlm.nih.gov/pubmed/14536075'>  Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA+ ClpXP Protease, D. Wah et al, Molecular Cell, Vol. 12, 355-363, August, 2003, PMID: 14536075  </a> </br> <a name=164>[16.4]</a> <a href='http://www.sciencedirect.com/science/article/pii/S0969212607003152'> Structure and Substrate Specifity of an SspB Ortholog: Desing Implications for AAA+ Adaptors, P. Chien et al, Cell Press, October 2007, 1296-1305, PMID: 17937918 </a> </br> <a name=165>[16.5]</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/16762842'> Engineering controllable protein degredation , McGinness et al, Mol Cell. 2006 Jun 9, PMID: 16762842 </a> </br> <a name=166>[16.6]</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/?term=Altered+Tethering+of+the+SspB+Adaptor+to+the+ClpXP+Protease+Causes+Changes+in+Substrate+Delivery'> Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinness et al, J Biol Chem. 2007 Apr 13; PMID: 17317664 </a> </br> <a name=167>[16.7]</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed'> http://www.ncbi.nlm.nih.gov/pubmed";
 +
content.type="Background";
 +
break;
case 17:
case 17:
 +
content.i = 17;
content.i = 17;
content.parents=[1];
content.parents=[1];
Line 197: Line 217:
break;
break;
-
case 18:
 
 +
case 18:
content.i = 18;
content.i = 18;
Line 210: Line 230:
break;
break;
-
case 19:
 
 +
case 19:
content.i = 19;
content.i = 19;
Line 225: Line 245:
case 20:
case 20:
-
 
content.i = 20;
content.i = 20;
Line 237: Line 256:
break;
break;
-
case 21:
 
 +
case 21:
content.i = 21;
content.i = 21;
Line 262: Line 281:
content.type="Background";  
content.type="Background";  
break;
break;
 +
case 33:
case 33:
Line 274: Line 294:
content.type="Background";
content.type="Background";
break;
break;
 +
case 34:
case 34:
 +
content.i = 34;
content.i = 34;
content.parents=[17];
content.parents=[17];
Line 288: Line 310:
case 36:
case 36:
-
 
content.i = 36;
content.i = 36;
Line 302: Line 323:
case 38:
case 38:
-
 
content.i = 38;
content.i = 38;
Line 315: Line 335:
-
case 45:
+
case 42:
 +
content.i = 42;
 +
content.parents=[74];
 +
content.childs=[43];
 +
content.titleShort = "sspB&alpha;";
 +
content.titleLong = "C. crescentus sspB&alpha;";
 +
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 [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-<sup>CC</sup>ssrA 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 <sup>CC</sup>ssrA, with an affinity being 175 times as large as for binding to <sup>EC</sup>ssrA (i.e. the E. coli ortholog).  By comparing the crystal structures of both sspB&alpha; and the compound of sspB&alpha; and <sup>CC</sup>ssrA, Chien et al. further proved that binding of sspB&alpha; to <sup>CC</sup>ssrA 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.type="Outlook";
 +
break;
 +
 +
 +
case 44:
 +
 +
content.i = 44;
 +
content.parents=[74];
 +
content.childs=[];
 +
content.titleShort = "ssrA";
 +
content.titleLong = "C. crescentus 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.text= "";
 +
content.type="Outlook";
 +
break;
 +
 +
 +
case 45:
content.i = 45;
content.i = 45;
Line 328: Line 373:
break;
break;
-
case 46:
 
 +
case 46:
content.i = 46;
content.i = 46;
Line 343: Line 388:
case 47:
case 47:
-
 
content.i = 47;
content.i = 47;
Line 357: Line 401:
case 48:
case 48:
-
 
content.i = 48;
content.i = 48;
Line 368: Line 411:
content.type="Background";
content.type="Background";
break;
break;
-
 
case 49:
case 49:
-
 
content.i = 49;
content.i = 49;
Line 383: Line 424:
content.type="Background";
content.type="Background";
break;
break;
-
 
case 50:
case 50:
-
 
content.i = 50;
content.i = 50;
Line 398: Line 437:
content.type="Background";
content.type="Background";
break;
break;
-
 
case 51:
case 51:
-
 
content.i = 51;
content.i = 51;
Line 413: Line 450:
content.type="Background";
content.type="Background";
break;
break;
-
 
case 53:
case 53:
-
 
content.i = 50;
content.i = 50;
Line 431: Line 466:
case 54:
case 54:
-
 
content.i = 54;
content.i = 54;
Line 444: Line 478:
 +
case 68:
-
 
-
case 44:
 
-
 
-
 
-
content.i = 44;
 
-
content.parents=[74];
 
-
content.childs=[];
 
-
content.titleShort = "ssrA";
 
-
content.titleLong = "C. crescentus 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.text= "";
 
-
content.type="Outlook";
 
-
break;
 
-
 
-
case 42:
 
-
 
-
 
-
content.i = 42;
 
-
content.parents=[74];
 
-
content.childs=[43];
 
-
content.titleShort = "sspB&alpha;";
 
-
content.titleLong = "C. crescentus sspB&alpha;";
 
-
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 [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-<sup>CC</sup>ssrA 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 <sup>CC</sup>ssrA, with an affinity being 175 times as large as for binding to <sup>EC</sup>ssrA (i.e. the E. coli ortholog).  By comparing the crystal structures of both sspB&alpha; and the compound of sspB&alpha; and <sup>CC</sup>ssrA, Chien et al. further proved that binding of sspB&alpha; to <sup>CC</sup>ssrA 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.type="Outlook";
 
-
break;
 
-
 
-
case 68:
 
content.i = 68;
content.i = 68;
content.parents=[54];
content.parents=[54];
Line 482: Line 489:
content.type="Background";
content.type="Background";
break;
break;
 +
case 71:
case 71:
 +
content.i = 71;
content.i = 71;
content.parents=[9];
content.parents=[9];
Line 496: Line 505:
case 72:
case 72:
 +
content.i = 72;
content.i = 72;
content.parents=[9];
content.parents=[9];
Line 508: Line 518:
case 73:
case 73:
 +
content.i = 73;
content.i = 73;
content.parents=[9];
content.parents=[9];
Line 518: Line 529:
break;
break;
-
case 74:
 
 +
case 74:
content.i = 74;
content.i = 74;
Line 531: Line 542:
break;
break;
-
case 137:
 
 +
case 137:
content.i = 137;  
content.i = 137;  
Line 543: Line 554:
content.type="Team";  
content.type="Team";  
break;
break;
 +
case 43:
case 43:
Line 555: Line 567:
  content.type="project";  
  content.type="project";  
  break;
  break;
 +
case 109:
case 109:
 +
content.i = 109;
content.i = 109;
content.parents=[105];
content.parents=[105];
Line 622: Line 636:
break;
break;
-
case 16:
+
 
-
content.i = 16;
+
-
content.parents=[12];
+
-
content.childs=[];
+
-
content.titleShort = "sspB-Split";
+
-
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.text= "The ClpXP protein degradation system can be used for inducible protein degradation as described by Davis et al.  They made use of the native ClpXP system in E. coli with a modified ssrA-tag (DAS+4) at the target protein. <sup><a href=#161>[16.1]</a></sup>  The modified DAS+4 ssrA cannot bind the ClpXP without SspB. <sup><a href=#165>[16.5]</a></sup> Using this dependency of the protein degradation on SspB, they decided to control the function of SspB by splitting it into two parts, each of which cannot induce degradation on its own. <sup><a href=#161>[16.1]</a></sup> </br> </br> Splitting of SspB is possible because its tripartite structure. It consists of (1.) a ssrA-tag binding and dimerization domain (SspB [CORE]), (2.) a flexible linker and (3.) a short peptide module that docks with ClpXP (SspB [XB]). <sup><a href=#163>[16.3]</a></sup> To test whether the linker length can be varied, the degradation rates of GFP-DAS+4 (0.3μM) with SspB (0.15μM) were tested in vitro with 4 different linker lengths (of 5, 25, 48 and 91 amino acids). The results showed that the 25 amino acids variant triggered the fastest degradation, followed by the 25 variant with 60%, 5 variant with 30% and 91 variant with 20% of the 25 variant rate. But even the 91 amino acids variant showed a 40times faster degradation rate than the degradation system without any SspB. <sup><a href=#166>[16.6]</a></sup>  Further experiments (e.g. the split system with FRB-FKBP12 as a linker, see below) demonstrated that even longer linker regions (more than 200 amino acids) are functional). Thus it can be concluded that not only the length of the linker is important but also its structure. <sup><a href=#161>[16.1]</a></sup> </br> </br> <img src='https://static.igem.org/mediawiki/2013/1/1f/Bonn-Gfp.jpg'> <sup><a href=#166>[16.6]</a></sup> </br> </br> To bring both SspB parts together again for inducible degradation, they were combined with a chemical inducible heterodimerisation system: FRB and FKBP12. <sup><a href=#161>[16.1]</a></sup> FKBP12 (FK 06 binding protein, 12 kDa) is a binding protein (108 amino acids <sup><a href=#167>[16.7]</a></sup>) for the small molecule rapamycin. FRB (FKBP-rapamycin binding domain) is the FKBP12-rapamycin binding domain (100 amino acids) of the mammalian protein mTor. In the absence of rapamycin, FKBP12 and FRB show no measurable interaction, while in the presence of rapamycin they build a strong FKBP-rapamycin-FRB ternary complex. <sup><a href=#162>[16.2]</a></sup> </br> </br> In order to achieve inducible degradation Davis et al. created the fusion proteins SspB[CORE]-FRB and FKBP12-SspB[XB]. SspB[CORE]-FRB interacts with the ssrA-tag of the target protein. FKBP12-SspB[XB] interacts with the ClpXP. In absence of rapamycin the two parts of SspB can only bind there particular targets but can’t interact with each other. Therefore, they don’t work as an adapter between the ssrA-taged protein and the ClpXP. By adding rapamycin FRB and FKBP12 dimerize. As a consequence the two parts of SspB get in a spatial closeness and function as an adapter. As a consequent the target protein gets degraded. <sup><a href=#161>[16.1]</a></sup> </br> </br> <img src='https://static.igem.org/mediawiki/2013/2/21/Bonn-rapa-split.jpg'> <sup><a href=#161>[16.1]</a></sup> </br> The efficiency of this system was demonstrated in the following in vitro experiments: GFP-DAS+4 was incubated with ClpXP, FKBP12-SspB[XP], SspB[CORE]-FRB and an ATP-regenerating system. Without rapamycin there was no degradation of GFP. The addition of rapamycin led to a reduction of GFP-ssrA of around 50% in only 360 seconds (degradation rate of 0.58min-1enzyme-1)(figure 2a). <sup><a href=#161>[16.1]</a></sup> Furthermore it was tested how long the degradation system needs to assemble and thus to reach the maximal degradation rate in this system. A time of 20 seconds was measured (figure 2b). <sup><a href=#161>[16.1]</a></sup> </br> </br> <img src='https://static.igem.org/mediawiki/2013/d/d0/Bonn-GFP-Abbau.jpg'> <sup><a href=#161>[16.1]</a></sup> </br> For in vivo testing they introduced the system into an SspB- mutant of E. coli by the plasmid pJD427. This plasmid contains SspB[CORE]-FRB with the weak constitutive promoter proB, FKBP12-SspB[XB] with the strong constitutive promoter proC and a medium-copy p15a origin of replication. For a target protein the lacI transcription repressor was used with a DAS+4-tag recombined to the C-terminus. Usually LacI represses lacZ transcription and thus production of β-galactosidase. Therefore degradation of LacI leads to an increasing β-galactosidase activity. As the assay showed absence of rapamycin results in no change of the β-galactosidase activity. Addition of rapamycin, however, leads to increasing β-galactosidase activity. Hence the system also worked in vivo (in an acceptable time). <sup><a href=#161>[16.1]</a></sup> </br> <img src='https://static.igem.org/mediawiki/2013/2/2d/Bonn-rapa-gel.jpg'> <sup><a href=#161>[16.1]</a></sup> </br> <h2> <b>References:</b> </h2> <a name=161>[16.1]</a> <a href=http://'www.ncbi.nlm.nih.gov/pmc/articles/PMC3220803/'> Small-Molecule Control of Protein Degradation Using Split Adaptors, J. Davis et al, ACS Chem. Biol. 2011, 6, 1205-1213, PMID: 21866931 </a> </br> <a name=162>[16.2]</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/15796538'> Charaterization of the FKBP•Rapamycin•FRB Ternary Complex, L. Banaszynski, C. Liu et al,  J. AM. CHEM. SOC. 2006, 127, 4715-4721, PMID: 15796538 </a> </br> <a name=163>[16.3]</a> <a href=http://'www.ncbi.nlm.nih.gov/pubmed/14536075'>  Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA+ ClpXP Protease, D. Wah et al, Molecular Cell, Vol. 12, 355-363, August, 2003, PMID: 14536075  </a> </br> <a name=164>[16.4]</a> <a href='http://www.sciencedirect.com/science/article/pii/S0969212607003152'> Structure and Substrate Specifity of an SspB Ortholog: Desing Implications for AAA+ Adaptors, P. Chien et al, Cell Press, October 2007, 1296-1305, PMID: 17937918 </a> </br> <a name=165>[16.5]</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/16762842'> Engineering controllable protein degredation , McGinness et al, Mol Cell. 2006 Jun 9, PMID: 16762842 </a> </br> <a name=166>[16.6]</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/?term=Altered+Tethering+of+the+SspB+Adaptor+to+the+ClpXP+Protease+Causes+Changes+in+Substrate+Delivery'> Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinness et al, J Biol Chem. 2007 Apr 13; PMID: 17317664 </a> </br> <a name=167>[16.7]</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed'> http://www.ncbi.nlm.nih.gov/pubmed";
+
-
content.type="Background";
+
-
break;
+
Line 745: Line 750:
content.i = 110;
content.i = 110;
-
 
content.parents=[100];
content.parents=[100];
-
 
content.childs=[];
content.childs=[];
-
 
content.titleShort = "LOV-Wars Shooter";
content.titleShort = "LOV-Wars Shooter";
-
 
content.titleLong = "LOV-Wars Shooter";
content.titleLong = "LOV-Wars Shooter";
-
 
content.summary= " In our effort to present our project in a simple and comprehensible way, we introduced already in may 2013 a java minigame into our wiki, which is a new approach in Human practice work, that has not been in iGEM before.";
content.summary= " In our effort to present our project in a simple and comprehensible way, we introduced already in may 2013 a java minigame into our wiki, which is a new approach in Human practice work, that has not been in iGEM before.";
-
 
content.text= " In our effort to present our project in a simple and comprehensible way, we introduced already in may 2013 a java minigame into our wiki, which is a new approach in Human practice work, that has not been in iGEM before. When people visit our wiki, they have the possibility to get to know central aspects of our project by playing and especially doing, which is generally a good way to learn.</br>The game is a mix of a simple shooter, where you need to hit things to raise your score, and an adventure game, where you have got to decide between different opportunities. At several score steps the player gets the possibility to achieve upgrades, that helps him to realize the central aspect of our project: The Degradation of specious proteins in bacteria.</br>We based the game on our introduced comic to intensify the connection to our Human practice work. The player might have already read the comic and, as connections are very important for understanding and learning, he is probably reminded to the different characters and their relations in the comic.</br>In the game the player starts at point zero in light induction: His UV laser gun kills any bacteria and therefore implements the simplest way to evoke something with light. The player will realize, that this laser may also unintentionally kill so called 'civilians' (bacteria without the targeted properties). Therefore he is able to upgrade his laser to a blue laser, which has got a defined wavelength and affects only the mechanism, that degrades special proteins. 'Civilians' are not disturbed by the blue laser. According to our comic so called 'clones' (bacteria, that have got the specious protein) will not be killed, but the specious protein is degraded. In our comic and our game 'evil clone warriors' are turned to likely, nice bacteria, when the 'evil' proteins in them are degraded.</br>In addition to that, 'Darth Cherry', the head of all clone warriors, appears at several steps in the game. You need to hit him several times until he dies/turns to a nice bacteria. The player can choose the 'plasmid of death' upgrade, which allows him to terminate Darth Cherry in one step. It shows the player, that you can change properties of bacteria by inserting plasmids and is surely an allusion to our kill-switch system.</br>Other upgrades show the player how to improve a system: You can get a wider and stronger laser to hit more bacteria, you can use nutrition to bait bacteria, you can use ice to make them slower. Surely all these upgrades are very generalized and simplified and do not totally correspond to reality, but our aim is not to present detailed information in the game. It just should create a base for people to understand our project, even if they are no experts in synthetic biology. Additionally the entertaining aspect of our game might attract internet-passersby, who are unintentionally informed about synthetic biology.";
content.text= " In our effort to present our project in a simple and comprehensible way, we introduced already in may 2013 a java minigame into our wiki, which is a new approach in Human practice work, that has not been in iGEM before. When people visit our wiki, they have the possibility to get to know central aspects of our project by playing and especially doing, which is generally a good way to learn.</br>The game is a mix of a simple shooter, where you need to hit things to raise your score, and an adventure game, where you have got to decide between different opportunities. At several score steps the player gets the possibility to achieve upgrades, that helps him to realize the central aspect of our project: The Degradation of specious proteins in bacteria.</br>We based the game on our introduced comic to intensify the connection to our Human practice work. The player might have already read the comic and, as connections are very important for understanding and learning, he is probably reminded to the different characters and their relations in the comic.</br>In the game the player starts at point zero in light induction: His UV laser gun kills any bacteria and therefore implements the simplest way to evoke something with light. The player will realize, that this laser may also unintentionally kill so called 'civilians' (bacteria without the targeted properties). Therefore he is able to upgrade his laser to a blue laser, which has got a defined wavelength and affects only the mechanism, that degrades special proteins. 'Civilians' are not disturbed by the blue laser. According to our comic so called 'clones' (bacteria, that have got the specious protein) will not be killed, but the specious protein is degraded. In our comic and our game 'evil clone warriors' are turned to likely, nice bacteria, when the 'evil' proteins in them are degraded.</br>In addition to that, 'Darth Cherry', the head of all clone warriors, appears at several steps in the game. You need to hit him several times until he dies/turns to a nice bacteria. The player can choose the 'plasmid of death' upgrade, which allows him to terminate Darth Cherry in one step. It shows the player, that you can change properties of bacteria by inserting plasmids and is surely an allusion to our kill-switch system.</br>Other upgrades show the player how to improve a system: You can get a wider and stronger laser to hit more bacteria, you can use nutrition to bait bacteria, you can use ice to make them slower. Surely all these upgrades are very generalized and simplified and do not totally correspond to reality, but our aim is not to present detailed information in the game. It just should create a base for people to understand our project, even if they are no experts in synthetic biology. Additionally the entertaining aspect of our game might attract internet-passersby, who are unintentionally informed about synthetic biology.";
-
 
content.type="Human Practice";
content.type="Human Practice";
break;
break;

Revision as of 14:25, 3 October 2013

Retrieved from "http://2013.igem.org/Template:Team:Bonn:NetworkData"