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content.text= "";

content.type="Background";

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break;

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case 16:

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content.i = 16;

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content.parents=[12];

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content.childs=[];

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content.titleShort = 'sspB-Split';

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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. <a href=#161>[16.1]</a> The modified DAS+4 ssrA cannot bind the ClpXP without SspB. <a href=#165>[16.5]</a> 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. <a href=#161>[16.1]</a> 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]). <a href=#163>[16.3]</a> 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. <a href=#166>[16.6]</a> 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. <a href=#161>[16.1]</a> <img src="https://static.igem.org/mediawiki/2013/1/1f/Bonn-Gfp.jpg"> <a href=#166>[16.6]</a> To bring both SspB parts together again for inducible degradation, they were combined with a chemical inducible heterodimerisation system: FRB and FKBP12. <a href=#161>[16.1]</a> FKBP12 (FK 06 binding protein, 12 kDa) is a binding protein (108 amino acids <a href=#167>[16.7]</a>) 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. <a href=#162>[16.2]</a> 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. <a href=#161>[16.1]</a> <img src="https://static.igem.org/mediawiki/2013/2/21/Bonn-rapa-split.jpg"> <a href=#161>[16.1]</a> 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). <a href=#161>[16.1]</a> 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). <a href=#161>[16.1]</a> <img src="https://static.igem.org/mediawiki/2013/d/d0/Bonn-GFP-Abbau.jpg"> <a href=#161>[16.1]</a> 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). <a href=#161>[16.1]</a> <img src="https://static.igem.org/mediawiki/2013/2/2d/Bonn-rapa-gel.jpg"> <a href=#161>[16.1]</a> <h2> References: </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> <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> <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> <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> <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> <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> <a name=167>[16.7]</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed> http://www.ncbi.nlm.nih.gov/pubmed';

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content.type='Background';

break;

@@ Line 213: / Line 213: @@
 content.text= "";
 content.type="Background";
+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;

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