Template:Team:Bonn:NetworkData

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.text= "<b>Introduction </b> </br> The sspB protein is an adaptor responsible for delivering ssrA-tagged substrates to the ClpXP protease in order to enhance their degradation.Thus, bacterias like E.coli or C.crescentus regulate the concentration of marked proteins and also are in control of their quality. Even though degeneration of tagged substrates is possible without sspB, sspB delivering is a common process, because it improves the affinity between ssrA and ClpXP^{<a href='#14.1'>14.1</a>}. </br> In our project, we used a sspB split variant instead of the normal sspB in order to control cleaving rate. The two parts of this version only stay divided until we ray them with light of a special wavelength. After that, the fractions form an unit and can function normal from now on as described below. </br> </br><b>Structure </b> </br>The sspB adaptor is a homomeric dimer (Fig. 1), which means that itconsists of two identical domains. Together the domains form a pore with the ssrA binding sites inside. Each domain also owns a C-terminal tail ending in a ClpX binding module, called XP. The amino-acid sequence of XP is highly conserved so that mutations in it mostly cause extremely decrease in activity, whereas the linker sequence differs from species to species ^{<a href='#14.2'>14.2</a>}^{<a href='#14.3'>14.3</a>}. <div align='left'><img src='https://static.igem.org/mediawiki/2013/5/5c/BonnsspB_Fig2.jpg' height='202' width='403'>Fig. 1: Ribbon diagramm of the sspB dimer with a bound srrA-tagged protein, from 'Versatile modes of peptide recognition by the AAA+ adaptor protein SspB, Levchenko et al, 2005, nature structural and molecular biology, PMID: 15880122</div> </br> </br></br></br><b>Function</b></br> SspB enhances degradation of ssrA-tagged proteins by lowering the K_M. Thus, with a given substrate concentration sspB-mediated cleaving runs faster than without sspB (Fig. 2) </br> <div align='left'><img src='https://static.igem.org/mediawiki/2013/d/d8/BonnSspB_Fig1.jpg' height='232' width='371'>Fig. 2: Diagramm, shows the degradation rate of GFP-ssrA without sspB, with sspB and with two mutations, the given substrate concentration is 0.3 &my;M, from 'Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA ClpXP Protease, Wah et al, Molecular Cell, 2003, PMID: 14536075' </div></br> Therefor, the sspB dimer contains a pore and while 'AADENY' is linked with the inside, the 'LAA'-domain (respectively 'DAS') faces outwards, free to bind ClpX (Fig. 3).</br> The affinity of this binding amounts around 20 &my;M, which suggests a relative strong  connection. The two extremely flexible ClpX binding tails with XP at the C-terminal end  dock on ClpX. So the 'LAA'- domain lies closely to ClpX's axial pore and can be bound to it. <div align='left'><img src='https://static.igem.org/mediawiki/2013/7/7c/BonnSsra_fig3.jpg' height='151' width='116'>Fig. 3: Model of sspB with a bound ssrA-tagged substrate, from 'Engineering controllable protein degradation, McGinness et al, Molecular cell, 2006, PMID:16762842' </div></br> To sum up, there are three bonds connecting the ssrA-sspB-ClpX-complex and making it relative stable: ssrA with sspB, sspB with ClpX and ssrA with ClpX. Hence follows a lower K_M than the direct binding process has^{<a href='#14.4'>14.4</a>}^{<a href='#14.5'>14.5</a>}. <h2> References </h2> <a id='14.1'>14.1</a>Bivalent Tethering of SspB to ClpXP Is Required for Efficient Substrate Delivery: A Protein-Design Study, Bolon et al, 2004, Molecular Cell, PMID: 14967151 </br><a id='14.2'>14.2</a>see above </br> <a id='14.3'>14.3</a> Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA ClpXP Protease, Wah et al, Molecular Cell, 2003, PMID: 14536075 </br> <a id='14.4'>14.4</a> see above</br> <a id='14.6'>[14.5]</a> Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </br> <a id='14.4'>14.4</a>  ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554</br>";  

content.text= "<b>Introduction </b> </br> The sspB protein is an adaptor responsible for delivering ssrA-tagged substrates to the ClpXP protease in order to enhance their degradation.Thus, bacterias like E.coli or C.crescentus regulate the concentration of marked proteins and also are in control of their quality. Even though degeneration of tagged substrates is possible without sspB, sspB delivering is a common process, because it improves the affinity between ssrA and ClpXP^{<a href='#14.1'>14.1</a>}. </br> In our project, we used a sspB split variant instead of the normal sspB in order to control cleaving rate. The two parts of this version only stay divided until we ray them with light of a special wavelength. After that, the fractions form an unit and can function normal from now on as described below. </br> </br><b>Structure </b> </br>The sspB adaptor is a homomeric dimer (Fig. 1), which means that itconsists of two identical domains. Together the domains form a pore with the ssrA binding sites inside. Each domain also owns a C-terminal tail ending in a ClpX binding module, called XP. The amino-acid sequence of XP is highly conserved so that mutations in it mostly cause extremely decrease in activity, whereas the linker sequence differs from species to species ^{<a href='#14.2'>14.2</a>}^{<a href='#14.3'>14.3</a>}. <div align='left'><img src='https://static.igem.org/mediawiki/2013/5/5c/BonnsspB_Fig2.jpg' height='202' width='403'>Fig. 1: Ribbon diagramm of the sspB dimer with a bound srrA-tagged protein, from 'Versatile modes of peptide recognition by the AAA+ adaptor protein SspB, Levchenko et al, 2005, nature structural and molecular biology, PMID: 15880122</div> </br> </br></br></br><b>Function</b></br> SspB enhances degradation of ssrA-tagged proteins by lowering the K_M. Thus, with a given substrate concentration sspB-mediated cleaving runs faster than without sspB (Fig. 2) </br> <div align='left'><img src='https://static.igem.org/mediawiki/2013/d/d8/BonnSspB_Fig1.jpg' height='232' width='371'>Fig. 2: Diagramm, shows the degradation rate of GFP-ssrA without sspB, with sspB and with two mutations, the given substrate concentration is 0.3 &my;M, from 'Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA ClpXP Protease, Wah et al, Molecular Cell, 2003, PMID: 14536075' </div></br> Therefor, the sspB dimer contains a pore and while 'AADENY' is linked with the inside, the 'LAA'-domain (respectively 'DAS') faces outwards, free to bind ClpX (Fig. 3).</br> The affinity of this binding amounts around 20 &my;M, which suggests a relative strong  connection. The two extremely flexible ClpX binding tails with XP at the C-terminal end  dock on ClpX. So the 'LAA'- domain lies closely to ClpX's axial pore and can be bound to it. <div align='left'><img src='https://static.igem.org/mediawiki/2013/7/7c/BonnSsra_fig3.jpg' height='151' width='116'>Fig. 3: Model of sspB with a bound ssrA-tagged substrate, from 'Engineering controllable protein degradation, McGinness et al, Molecular cell, 2006, PMID:16762842' </div></br> To sum up, there are three bonds connecting the ssrA-sspB-ClpX-complex and making it relative stable: ssrA with sspB, sspB with ClpX and ssrA with ClpX. Hence follows a lower K_M than the direct binding process has^{<a href='#14.4'>14.4</a>}^{<a href='#14.5'>14.5</a>}. <h2> References </h2> <a id='14.1'>14.1</a>Bivalent Tethering of SspB to ClpXP Is Required for Efficient Substrate Delivery: A Protein-Design Study, Bolon et al, 2004, Molecular Cell, PMID: 14967151 </br><a id='14.2'>14.2</a>see above </br> <a id='14.3'>14.3</a> Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA ClpXP Protease, Wah et al, Molecular Cell, 2003, PMID: 14536075 </br> <a id='14.4'>14.4</a> see above</br> <a id='14.6'>[14.5]</a> Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </br> <a id='14.4'>14.4</a>  ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554</br>";  

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.text= "<b> Introduction </b> </br> For cells it is important to have a steady control over their own functions and reactions. During evolution many regulation systems evolved with controlling protein concentrations amongst them. In fact, increasing or decreasing protein amount is an effective way to manipulate cell activities. Therefore proteins can be marked with special tag sequences, which allows proteolytic enzymes to degrade them. One of those tags is called ssrA and relates to proteases like the ClpXP  complex in E.coli. The tag also allows adaptor proteins such as sspB binding and delivering substrates to the proteases in order to make the process more efficient^{<a href='#15.1'>15.1</a>}. </br> For our project, the ssrA tag was very important, because we synthetically marked proteins with it to degrade them by placing the ssrA gen-code next to the protein code and letting ribosomes translate  the new sequence. We also used sspB and the adaptor-mediated variant as described below, whereas the direct binding pathway wasn't an opinion for us. The reason is that we needed to control degradation level and therefor we set in a split version of sspB, which we could reunite through light radiation. For further information about the ClpXP degradation system in our project go to ClpXP general. Although it was not part of our project, the information in chapter &quot;Translation control&quot; exhiit another important functional aspect of ssrA tags.</br> </br><b>Structure </b></br>The ssrA tag is a short sequence consisting of eleven amino-acids and is translated with the associated protein simultaneously. The sequence can be divided into two functional parts (Fig. 1). The &quot;AANDENY&quot;-part, which is directly connected with the C-terminal end of the protein, is responsible for the binding to the sspB adaptor. Each letter in the part name stands for another amino-acid, A for example means Alanine. The other part, called &quot;LAA&quot;, interacts with the ClpX subunit of the ClpXP protease. The parts are connected over an Alanine molecule^{<a href='#15.2'>15.2</a>}. </br> </br><div align='left'><img src='https://static.igem.org/mediawiki/2013/1/18/BonnSsra_fig1.jpg' height='76' width='344'>Fig. 1: amino-acid sequence of ssrA, from &quot;Engineering controllable protein degradation, McGinness et al, Molecular cell, 2006, PMID:16762842&quot; </div></br><b>Function </b> </br>The ssrA tag works as a degrading signal for proteases like ClpXP. Therefor ClpX owns a ssrA binding site at its axial pore. According to the availability of sspB adaptors, there are two different binding pathways. </br> </br>1. Direct binding: If a tagged protein and the ClpX subunit incidentally bump into each other in correct orientation, they develop a binding. The binding site of ClpX is made out of several loops and ssrA can be crosslinked to them. The determinant factor for this binding is the negative charged &alpha;-COOH group on the terminal alanine of ssrA, because the loops are positive charged. Using this way, ClpXP reaches a maximum degradation rate of around 4 proteins per minute^{<a href='#15.3'>15.3</a>}.</br> </br><div align='left'><img src='https://static.igem.org/mediawiki/2013/a/a7/BonnSsra_fig2.jpg' height='281' width='361'>Fig. 2: direct binding of a tagged GFP protein, GFP is an green fluorescence protein, from &quot;Protein unfolding by a AAA+ protease is dependent on ATP-hydrolysis rates and substrate energy landscapes, Martin et al, Nature Structural & Molecular Biology, 2008, PMID: 18223658&quot;</div> </br>2. Adaptor-mediated binding: sspB is an adaptor protein with a special binding site for the &quot;AADENY&quot;-domain of ssrA. Therefor, the sspB dimer contains a pore in each subunit and while &quot;AADENY&quot; is linked with the inside, the &quot;LAA&quot;-domain faces outwards, free to bind ClpX (Fig. 2). The affinity of this binding amounts around 20 &my;M, which suggests a relative strong  connection. The sspB dimer also owns two extremely flexible ClpX binding tails at each C-terminal end. With docking on ClpX, the &quot;LAA&quot;-domain lies closely to ClpX's axial pore and can be bound to it.To sum up, there are three bonds connecting the ssrA-sspB-ClpX-complex and making it relative stable: ssrA with sspB, sspB with ClpX and ssrA with ClpX. Hence follows a lower K_M than the direct binding process has (hab hierzu keine konkreten Daten). This lower K_M means, that a smaller amount of substrate are needed to reach the maximum degradation speed. So actually sspB doesn't increase the maximum speed, but this tempo can be reached with less substrate concentrations^{<a href='#15.4'>15.4</a>}^{<a href='#15.5'>15.5</a>}.</br> </br><div align='left'><img src='https://static.igem.org/mediawiki/2013/7/7c/BonnSsra_fig3.jpg' height='251' width='216'>Fig. 3: ssrA tag with sspB adaptor and protein substrate, from &quot;Engineering controllable protein degradation, McGinness et al, Molecular cell, 2006, PMID:16762842&quot; </div> </br><b>Translation control</b> </br>Beneath the already mentioned functions, ssrA tags also play a role in quality control during ribosomal translation of genetic codes into protein sequences. In a normal working translation tRNA molecules deliver the amino-acids and a ribosome puts them together in the right order, using a mRNA strand as template. But this complicated process can be afflicted with mistakes, for example premature abruption or missing stop codons. Mistakes mostly result in defect proteins, which can be dangerous for he cell. In order to circumvent this danger, defect proteins are tagged with ssrA for quick degradation by special tmRNA molecules. TmRNA is a mixture of mRNA and tRNA. On the one hand it is formed like a tRNA molecule, is able to bind to a ribosome and delivers one amino-acid, but on the other hand it do not have an anticodon. Instead, an ORF mRNA part can be found. ORF means &quot;open reading frame&quot; and is a coding sequence mostly for degradation tags like ssrA. As shown in figure 5, ssrA assembly is complex process. The tmRNA molecule binds to the A site of a stalled ribosome, takes over the already assembled amino-acid sequence and adds Alanine. Then it swaps the template mRNA for its ORF region and finishes translation with the new template. As a result the defect protein is now tagged and can be degradated by proteases like ClpXP^{<a href='#15.6'>15.6</a>}.</br> </br><div align='left'><img src='https://static.igem.org/mediawiki/2013/0/02/BonnSsra_fig4.jpg' height='500' width='325'>Fig. 4: Model for tmRNA-mediated tagging, from &quot;The tmRNA System for Translational Surveillance and Ribosome Rescue, Moore SD et al, Annual Reviews Biochemistry, 2007, PMID: 17291191&quot;</div> </br><h2><b> References </b></h2> </br><a id='15.1'>15.1</a> Engineering controllable protein degradation, McGinnes KE et al, Molecular cell, 2006, PMID: 16762842<a id='15.2'>[15.2]</a>  Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </br><a id='15.3'>15.3</a> ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554</br><a id='15.4'>15.4</a>  See above </br> <a id='15.5'>15.5</a>  Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </br><a id='15.6'>15.6</a> The tmRNA System for Translational Surveillance and Ribosome Rescue, Moore SD et al, Annual Reviews Biochemistry, 2007, PMID: 17291191</br>";

content.text= "<b> Introduction </b> </br> For cells it is important to have a steady control over their own functions and reactions. During evolution many regulation systems evolved with controlling protein concentrations amongst them. In fact, increasing or decreasing protein amount is an effective way to manipulate cell activities. Therefore proteins can be marked with special tag sequences, which allows proteolytic enzymes to degrade them. One of those tags is called ssrA and relates to proteases like the ClpXP  complex in E.coli. The tag also allows adaptor proteins such as sspB binding and delivering substrates to the proteases in order to make the process more efficient^{<a href='#15.1'>15.1</a>}. </br> For our project, the ssrA tag was very important, because we synthetically marked proteins with it to degrade them by placing the ssrA gen-code next to the protein code and letting ribosomes translate  the new sequence. We also used sspB and the adaptor-mediated variant as described below, whereas the direct binding pathway wasn't an opinion for us. The reason is that we needed to control degradation level and therefor we set in a split version of sspB, which we could reunite through light radiation. For further information about the ClpXP degradation system in our project go to ClpXP general. Although it was not part of our project, the information in chapter &quot;Translation control&quot; exhiit another important functional aspect of ssrA tags.</br> </br><b>Structure </b></br>The ssrA tag is a short sequence consisting of eleven amino-acids and is translated with the associated protein simultaneously. The sequence can be divided into two functional parts (Fig. 1). The &quot;AANDENY&quot;-part, which is directly connected with the C-terminal end of the protein, is responsible for the binding to the sspB adaptor. Each letter in the part name stands for another amino-acid, A for example means Alanine. The other part, called &quot;LAA&quot;, interacts with the ClpX subunit of the ClpXP protease. The parts are connected over an Alanine molecule^{<a href='#15.2'>15.2</a>}. </br> </br><div align='left'><img src='https://static.igem.org/mediawiki/2013/1/18/BonnSsra_fig1.jpg' height='76' width='344'>Fig. 1: amino-acid sequence of ssrA, from &quot;Engineering controllable protein degradation, McGinness et al, Molecular cell, 2006, PMID:16762842&quot; </div></br><b>Function </b> </br>The ssrA tag works as a degrading signal for proteases like ClpXP. Therefor ClpX owns a ssrA binding site at its axial pore. According to the availability of sspB adaptors, there are two different binding pathways. </br> </br>1. Direct binding: If a tagged protein and the ClpX subunit incidentally bump into each other in correct orientation, they develop a binding. The binding site of ClpX is made out of several loops and ssrA can be crosslinked to them. The determinant factor for this binding is the negative charged &alpha;-COOH group on the terminal alanine of ssrA, because the loops are positive charged. Using this way, ClpXP reaches a maximum degradation rate of around 4 proteins per minute^{<a href='#15.3'>15.3</a>}.</br> </br><div align='left'><img src='https://static.igem.org/mediawiki/2013/a/a7/BonnSsra_fig2.jpg' height='281' width='361'>Fig. 2: direct binding of a tagged GFP protein, GFP is an green fluorescence protein, from &quot;Protein unfolding by a AAA+ protease is dependent on ATP-hydrolysis rates and substrate energy landscapes, Martin et al, Nature Structural & Molecular Biology, 2008, PMID: 18223658&quot;</div> </br>2. Adaptor-mediated binding: sspB is an adaptor protein with a special binding site for the &quot;AADENY&quot;-domain of ssrA. Therefor, the sspB dimer contains a pore in each subunit and while &quot;AADENY&quot; is linked with the inside, the &quot;LAA&quot;-domain faces outwards, free to bind ClpX (Fig. 2). The affinity of this binding amounts around 20 &my;M, which suggests a relative strong  connection. The sspB dimer also owns two extremely flexible ClpX binding tails at each C-terminal end. With docking on ClpX, the &quot;LAA&quot;-domain lies closely to ClpX's axial pore and can be bound to it.To sum up, there are three bonds connecting the ssrA-sspB-ClpX-complex and making it relative stable: ssrA with sspB, sspB with ClpX and ssrA with ClpX. Hence follows a lower K_M than the direct binding process has (hab hierzu keine konkreten Daten). This lower K_M means, that a smaller amount of substrate are needed to reach the maximum degradation speed. So actually sspB doesn't increase the maximum speed, but this tempo can be reached with less substrate concentrations^{<a href='#15.4'>15.4</a>}^{<a href='#15.5'>15.5</a>}.</br> </br><div align='left'><img src='https://static.igem.org/mediawiki/2013/7/7c/BonnSsra_fig3.jpg' height='251' width='216'>Fig. 3: ssrA tag with sspB adaptor and protein substrate, from &quot;Engineering controllable protein degradation, McGinness et al, Molecular cell, 2006, PMID:16762842&quot; </div> </br><b>Translation control</b> </br>Beneath the already mentioned functions, ssrA tags also play a role in quality control during ribosomal translation of genetic codes into protein sequences. In a normal working translation tRNA molecules deliver the amino-acids and a ribosome puts them together in the right order, using a mRNA strand as template. But this complicated process can be afflicted with mistakes, for example premature abruption or missing stop codons. Mistakes mostly result in defect proteins, which can be dangerous for he cell. In order to circumvent this danger, defect proteins are tagged with ssrA for quick degradation by special tmRNA molecules. TmRNA is a mixture of mRNA and tRNA. On the one hand it is formed like a tRNA molecule, is able to bind to a ribosome and delivers one amino-acid, but on the other hand it do not have an anticodon. Instead, an ORF mRNA part can be found. ORF means &quot;open reading frame&quot; and is a coding sequence mostly for degradation tags like ssrA. As shown in figure 5, ssrA assembly is complex process. The tmRNA molecule binds to the A site of a stalled ribosome, takes over the already assembled amino-acid sequence and adds Alanine. Then it swaps the template mRNA for its ORF region and finishes translation with the new template. As a result the defect protein is now tagged and can be degradated by proteases like ClpXP^{<a href='#15.6'>15.6</a>}.</br> </br><div align='left'><img src='https://static.igem.org/mediawiki/2013/0/02/BonnSsra_fig4.jpg' height='500' width='325'>Fig. 4: Model for tmRNA-mediated tagging, from &quot;The tmRNA System for Translational Surveillance and Ribosome Rescue, Moore SD et al, Annual Reviews Biochemistry, 2007, PMID: 17291191&quot;</div> </br><h2><b> References </b></h2> </br><a id='15.1'>15.1</a> Engineering controllable protein degradation, McGinnes KE et al, Molecular cell, 2006, PMID: 16762842<a id='15.2'>[15.2]</a>  Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </br><a id='15.3'>15.3</a> ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554</br><a id='15.4'>15.4</a>  See above </br> <a id='15.5'>15.5</a>  Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </br><a id='15.6'>15.6</a> The tmRNA System for Translational Surveillance and Ribosome Rescue, Moore SD et al, Annual Reviews Biochemistry, 2007, PMID: 17291191</br>";

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.";