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content.titleLong = "Ec. SspB adaptor"; | content.titleLong = "Ec. SspB adaptor"; | ||
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<sup><a href='#14.1'>14.1</a></sup>. </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 <sup><a href='#14.2'>14.2</a></sup><sup><a href='#14.3'>14.3</a></sup>. <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<sub>M</sub>. 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<sub>M</sub> than the direct binding process has<sup><a href='#14.4'>14.4</a></sup><sup><a href='#14.5'>14.5</a></sup>. <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<sup><a href='#14.1'>14.1</a></sup>. </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 <sup><a href='#14.2'>14.2</a></sup><sup><a href='#14.3'>14.3</a></sup>. <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<sub>M</sub>. 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<sub>M</sub> than the direct binding process has<sup><a href='#14.4'>14.4</a></sup><sup><a href='#14.5'>14.5</a></sup>. <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.type="Background"; | content.type="Background"; | ||
break; | break; | ||
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content.childs=[41,74,43]; | content.childs=[41,74,43]; | ||
content.titleShort = "C. crescentus"; | content.titleShort = "C. crescentus"; | ||
| - | content.titleLong = " | + | content.titleLong = "Ortholog ClpXP system in C. crescentus"; |
| - | content.summary= " | + | content.summary= "Using the ortholog ClpXP protein degradation system of C. crescentus in E. coli enables us to induce protein degradation in native E. coli without interfering with their natural system."; |
content.text= "Caulobacter crescentus is a Gram-negative α-protobacterium often found in fresh water lakes or in the sea. Its cell division cycle is a favoured object of study due to its remarkable asymmetry. <sup><a href=401>40.1</a></sup></br>ClpXP is a protease that degrades proteins tagged with the ssrA peptide. sspBα is an adaptor protein that can accelerate protein degradation by tethering ssrA-tagged proteins towards the ClpX subunit of the ClpXP protease.</br>For further information, please browse the related articles. </br></br><h2>References</h2></br><a href=401>40.1</a><a href=’ http://www.amazon.com/Brock-Biology-Microorganisms-13th-Edition/dp/032164963X’> Brock Microbiology of Microorganisms, Madigan et al., Pearson, German edition Vol. 13, 2012</a>"; | content.text= "Caulobacter crescentus is a Gram-negative α-protobacterium often found in fresh water lakes or in the sea. Its cell division cycle is a favoured object of study due to its remarkable asymmetry. <sup><a href=401>40.1</a></sup></br>ClpXP is a protease that degrades proteins tagged with the ssrA peptide. sspBα is an adaptor protein that can accelerate protein degradation by tethering ssrA-tagged proteins towards the ClpX subunit of the ClpXP protease.</br>For further information, please browse the related articles. </br></br><h2>References</h2></br><a href=401>40.1</a><a href=’ http://www.amazon.com/Brock-Biology-Microorganisms-13th-Edition/dp/032164963X’> Brock Microbiology of Microorganisms, Madigan et al., Pearson, German edition Vol. 13, 2012</a>"; | ||
content.type="Project"; | content.type="Project"; | ||
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content.parents=[40]; | content.parents=[40]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "<sup>Cc</sup>ClpXP"; |
| - | content.titleLong = "C. crescentus ClpXP"; | + | content.titleLong = "C. crescentus: ClpXP"; |
content.summary= "This article is about the ClpXP protease system in C. crescentus and regulation of proteolysis via ClpXP. It also provides information about similarities and differences between C. crescentus and E. coli orthologs of related proteins."; | content.summary= "This article is about the ClpXP protease system in C. crescentus and regulation of proteolysis via ClpXP. It also provides information about similarities and differences between C. crescentus and E. coli orthologs of related proteins."; | ||
content.text= "<div class='content-image'><img src='https://static.igem.org/mediawiki/2013/d/de/Bonn-41-CC-ClpXP.png'>ClpXP mediated proteolysis <sup><a href=#413>41.3</a></sup></div>ClpXP is an AAA+ protease which is of particular importance for proper cell-cycle progression. It consists of a hexameric ClpX subunit, which recognizes and unfolds tagged proteins, while ATP is hydrolyzed, and a ClpP subunit, that contains the actual peptidase domain. <sup><a href=#411>41.1</a>, <a href=#412>41.2</a></sup> Its structure is highly conserved, such that E. coli and C. crescentus orthologs are very similar. </br>Specific activity of the ClpXP protease is mediated by sspB and ssrA. ssrA is a short peptide consisting of fourteen amino acids in C. crescentus. Proteins which need to be degraded, e.g. for regulatory purpose or due to errors in their structure, are tagged with the ssrA peptide that can be recognized by the ClpX subunit. sspB is a dimeric adaptor protein in C. crescentus which tethers ssrA-tagged proteins to the ClpXP protease and, in this way, accelerates protein degradation. </br>Whilst sspB protein structure is well- conserved among many microorganisms, the structure of <sup>CC</sup>sspBα (i.e., the C. crescentus ortholog of sspB) shows significant differences compared to other orthologs. For example, <sup>CC</sup>sspBα and <sup>EC</sup>sspB (i.e., the E. coli ortholog) only show up with sequence identities of 16%, while CCsspB is still able to specifically bind to <sup>EC</sup>ClpXP<sup><a href=#412>41.2</a></sup>. SsrA tags can also be very different among microorganismic species. </br>This makes it possible to establish a protein degradation system in E. coli involving <sup>CC</sup>sspBα and <sup>EC</sup>ClpXP, as long as the ssrA tag can be recognized by both <sup>CC</sup>sspBα and <sup>EC</sup>ClpXP. </br></br><h2>References</h2></br><a name=411>41.1</a> <a href=’ http://www.ncbi.nlm.nih.gov/pubmed/17937918’>Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a></br><a name=412>41.2</a> <a href=’http://www.ncbi.nlm.nih.gov/pubmed/20014030’>Versatile modes of peptide recognition by the ClpX N domain mediate alternative adaptor-binding specificities in different bacterial species, Chowdhury et al., Protein Science, 2010, PMID: 20014030</a></br><a name=413>41.3</a> <a href='http://www.biochem.umass.edu/faculty/peter-chien'>Peter Chien, Department of Biochemistry and Molecular Biology (official website), http://www.biochem.umass.edu/faculty/peter-chien, picture URL: cchttp://www.biochem.umass.edu/sites/biochem/files/resize/ClpX-300x268.jpg</a>"; | content.text= "<div class='content-image'><img src='https://static.igem.org/mediawiki/2013/d/de/Bonn-41-CC-ClpXP.png'>ClpXP mediated proteolysis <sup><a href=#413>41.3</a></sup></div>ClpXP is an AAA+ protease which is of particular importance for proper cell-cycle progression. It consists of a hexameric ClpX subunit, which recognizes and unfolds tagged proteins, while ATP is hydrolyzed, and a ClpP subunit, that contains the actual peptidase domain. <sup><a href=#411>41.1</a>, <a href=#412>41.2</a></sup> Its structure is highly conserved, such that E. coli and C. crescentus orthologs are very similar. </br>Specific activity of the ClpXP protease is mediated by sspB and ssrA. ssrA is a short peptide consisting of fourteen amino acids in C. crescentus. Proteins which need to be degraded, e.g. for regulatory purpose or due to errors in their structure, are tagged with the ssrA peptide that can be recognized by the ClpX subunit. sspB is a dimeric adaptor protein in C. crescentus which tethers ssrA-tagged proteins to the ClpXP protease and, in this way, accelerates protein degradation. </br>Whilst sspB protein structure is well- conserved among many microorganisms, the structure of <sup>CC</sup>sspBα (i.e., the C. crescentus ortholog of sspB) shows significant differences compared to other orthologs. For example, <sup>CC</sup>sspBα and <sup>EC</sup>sspB (i.e., the E. coli ortholog) only show up with sequence identities of 16%, while CCsspB is still able to specifically bind to <sup>EC</sup>ClpXP<sup><a href=#412>41.2</a></sup>. SsrA tags can also be very different among microorganismic species. </br>This makes it possible to establish a protein degradation system in E. coli involving <sup>CC</sup>sspBα and <sup>EC</sup>ClpXP, as long as the ssrA tag can be recognized by both <sup>CC</sup>sspBα and <sup>EC</sup>ClpXP. </br></br><h2>References</h2></br><a name=411>41.1</a> <a href=’ http://www.ncbi.nlm.nih.gov/pubmed/17937918’>Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a></br><a name=412>41.2</a> <a href=’http://www.ncbi.nlm.nih.gov/pubmed/20014030’>Versatile modes of peptide recognition by the ClpX N domain mediate alternative adaptor-binding specificities in different bacterial species, Chowdhury et al., Protein Science, 2010, PMID: 20014030</a></br><a name=413>41.3</a> <a href='http://www.biochem.umass.edu/faculty/peter-chien'>Peter Chien, Department of Biochemistry and Molecular Biology (official website), http://www.biochem.umass.edu/faculty/peter-chien, picture URL: cchttp://www.biochem.umass.edu/sites/biochem/files/resize/ClpX-300x268.jpg</a>"; | ||
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content.parents=[74]; | content.parents=[74]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "<sup>Cc</sup>sspBα"; |
| - | content.titleLong = "C. crescentus sspBα"; | + | content.titleLong = "C. crescentus: sspBα"; |
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 <sup><a href=#421>42.1</a></sup></div>The sspBα dimeric structure is stabilized by two α-helices in interaction, as part B of the figure above shows, each of them located at the N-terminus of either sspBα molecule. The subsequent parts of the protein form a domain consisting of two β-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. <sup><a href=#422>42.2</a></sup> </br>Chien et al. <sup><a href=#421>42.1</a></sup> compared crystal structures of C. crescentus sspBα and its E. coli and H. influenzae sspB orthologs, discovering that in sspBα the α-helices are significantly longer, more twisted and cover a larger cross section area than the other two sspB orthologs. Also considering that β-sheets are rotated by around 20° 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 sspBα dimer in C. crescentus, while they are parallel in γ-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 sspBα added, (2) with mutated sspBα(Q74A) added , (3) with wildtype sspBα added can be visualized. <sup><a href=#421>42.1</a></sup></div>Chien et al. point out that although there are the remarkable differences in protein structure between sspBα and its γ-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 sspBα binding to the protein which needs to be tethered to the ClpXP protease strongly depends on which ssrA ortholog the protein is tagged with. sspBα 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 sspBα and the compound of sspBα and <sup>CC</sup>ssrA, Chien et al. further proved that binding of sspBα to <sup>CC</sup>ssrA does not lead to significant changes of its 3D conformation.</br></br><h2>References</h2></br><a name=421>42.1</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/17937918'>Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a></br><a name=422>42.2</a><a href='http://www.ncbi.nlm.nih.gov/pubmed/14967151'> 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</a>"; | 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 <sup><a href=#421>42.1</a></sup></div>The sspBα dimeric structure is stabilized by two α-helices in interaction, as part B of the figure above shows, each of them located at the N-terminus of either sspBα molecule. The subsequent parts of the protein form a domain consisting of two β-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. <sup><a href=#422>42.2</a></sup> </br>Chien et al. <sup><a href=#421>42.1</a></sup> compared crystal structures of C. crescentus sspBα and its E. coli and H. influenzae sspB orthologs, discovering that in sspBα the α-helices are significantly longer, more twisted and cover a larger cross section area than the other two sspB orthologs. Also considering that β-sheets are rotated by around 20° 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 sspBα dimer in C. crescentus, while they are parallel in γ-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 sspBα added, (2) with mutated sspBα(Q74A) added , (3) with wildtype sspBα added can be visualized. <sup><a href=#421>42.1</a></sup></div>Chien et al. point out that although there are the remarkable differences in protein structure between sspBα and its γ-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 sspBα binding to the protein which needs to be tethered to the ClpXP protease strongly depends on which ssrA ortholog the protein is tagged with. sspBα 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 sspBα and the compound of sspBα and <sup>CC</sup>ssrA, Chien et al. further proved that binding of sspBα to <sup>CC</sup>ssrA does not lead to significant changes of its 3D conformation.</br></br><h2>References</h2></br><a name=421>42.1</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/17937918'>Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a></br><a name=422>42.2</a><a href='http://www.ncbi.nlm.nih.gov/pubmed/14967151'> 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</a>"; | ||
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content.parents=[40]; | content.parents=[40]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "<sup>Cc</sup>sspB Split"; |
| - | content.titleLong = " | + | content.titleLong = "sspB Split in C. crescentus"; |
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 <sup><a href=#431>43.1</a></sup>. Thus ccssrA only binds ccSspB but not E. coli SspB. <sup><a href=#432>43.2</a></sup> <sup><a href=#433>43.3</a></sup> <sup><a href=#434>43.4</a></sup> 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. <sup><a href=#431>43.1</a></sup> </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 <sup><a href=#431>43.1</a></sup>. </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'> <sup><a href=#431>43.1</a></sup> <h2>References:</h2> </br> <a name=431>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=432>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=433>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=434>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 <sup><a href=#431>43.1</a></sup>. Thus ccssrA only binds ccSspB but not E. coli SspB. <sup><a href=#432>43.2</a></sup> <sup><a href=#433>43.3</a></sup> <sup><a href=#434>43.4</a></sup> 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. <sup><a href=#431>43.1</a></sup> </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 <sup><a href=#431>43.1</a></sup>. </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'> <sup><a href=#431>43.1</a></sup> <h2>References:</h2> </br> <a name=431>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=432>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=433>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=434>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>"; | ||
| Line 403: | Line 403: | ||
content.parents=[74]; | content.parents=[74]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = "SsrA"; | + | content.titleShort = "<sup>Cc</sup>SsrA"; |
content.titleLong = "C. crescentus ssrA and its application in E. coli"; | content.titleLong = "C. crescentus ssrA and its application in E. coli"; | ||
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."; | ||
| Line 587: | Line 587: | ||
content.childs=[]; | content.childs=[]; | ||
content.titleShort = "MazEF"; | content.titleShort = "MazEF"; | ||
| - | content.titleLong = "A | + | content.titleLong = "A Killswitch using the toxin-antitoxin module MazEF in E. coli"; |
content.summary= "The toxin-antitoxin system MazEF is composed by an upstream gene <i>mazE</i>, encoding a labile antitoxin, and a downstream gene <i>mazF</i>, that encodes a stable toxin. Connecting our light inducible protein degradation system to the antitoxin MazE allows light inducible cell death, as predominance of MazF in a bacterium activates cell death pathway."; | content.summary= "The toxin-antitoxin system MazEF is composed by an upstream gene <i>mazE</i>, encoding a labile antitoxin, and a downstream gene <i>mazF</i>, that encodes a stable toxin. Connecting our light inducible protein degradation system to the antitoxin MazE allows light inducible cell death, as predominance of MazF in a bacterium activates cell death pathway."; | ||
content.text= "Our system of light inducible protein degradation can be utilized to degrade any specific protein and is therefore usable to realize a light inducible kill-switch system. A connection of protein degradation to a cell death pathway is represented by the stress-induced toxin-antitoxin module <i>mazEF</i> in Escherichia coli. <i>mazEF</i> is located on the chromosome in E.coli which is associated with programmed cell death. The toxin-antitoxin system is composed by an upstream gene <i>mazE</i>, encoding a labile antitoxin, and a downstream gene mazF, that encodes a stable toxin. </br>The product of <i>mazF</i> cleaves mRNAs and tmRNAs at a specific site, which leads to an inhibition of translation. MazF shows a specific cleaving mechanism, which is not well understood yet, but shows that there is also protein synthesis which is unaffected by MazF. These proteins are presumably part of a cell death pathway. </br>The effect of <i>mazF</i> is suppressed by the Product of <i>mazE</i> which is degraded by the Protease ClpAP in bacteria. As a result of stressful conditions expression of the chromosomal <i>mazEF</i> module is reduced which leads to an imbalance between the products of <i>mazF</i> and <i>mazE</i>: When expression is lowered the stable toxin of <i>mazF</i> still persists while the labile antitoxin of <i>mazE</i> is degraded and can no longer suppress the effect of <i>mazF</i> leading to acute toxicity and cell death. </br><i>MazEF</i>-mediated cell death in E. coli can be caused by:<ul><li>extreme amino acid starvation<sup><a href = #674>67.4</a></sup><sup><a href = #675>67.5</a></sup></li><li> inhibition of transcription and/or translation by antibiotics such as rifampin, chloramphenicol, and spectinomycin under specific growth conditions<sup><a href = #676>67.6</a></sup></li><li>inhibition of translation by the Doc protein of prophage P1<sup><a href = #677>67.7</a></sup></li><li>DNA damage caused by thymine starvation<sup><a href = #678>67.8</a></sup> as well as by mitomycin C, nalidixic acid, and UV irradiation<sup><a href = #679>67.9</a></sup></li><li>oxidative stress (H2O2)<sup><a href = #679'>67.9</a></sup></li></ul>Amitai et al. tested in 2004 the Hypothesis of Pedersen et al.<sup><a href = #672>67.2</a></sup>, that chromosomal toxin-antitoxin systems may rather cause a state of reversible bacteriostasis than programmed cell death<sup><a href = #671>67.1</a></sup>.Therefore E.coli strain MC4100 Δ<i>mazE</i>F relA1 lacIq was cotransformated with:<ul><li>pBad-<i>mazF</i></li><li>pQE-Δhis-<i>mazE</i></li></ul><i>mazF</i>-expression can be induced by the addition of Arabinose via the pBad promoter of the first plasmid. The transformation of the second plasmid results firstly in the repression of <i>mazE</i> expression, whereas when IPTG is added <i>mazE</i> production is induced.</br><div class='content-image' align='center' height=501 width=410><a href='https://static.igem.org/mediawiki/2013/a/ac/Team_Bonn_MazF_1.png'><img src='https://static.igem.org/mediawiki/2013/a/ac/Team_Bonn_MazF_1.png' height=491 width=400></a></br><i>Ability of E. coli cells that had been ectopically overexpressing MazF in liquid medium to form colonies when ectopically overexpressing MazE on plates. The cultures were grown in LB medium (A) or M9 minimal medium with 0.5% glycerol (B) at 37°C to midlogarithmic phase (OD600, 0.5)<sup><a href = #671>67.1</a></sup>.</i></div>Using these tools, Amitai et al. tested the effect of MazE overproduction on MazF-overproducing bacteria during growth in liquid medium.</br>The E.coli strain was incubated in LB medium. After <i>mazF</i> expression was induced by adding arabinose two samples were taken at several time points. To repress <i>mazF</i> expression to both of them glucose was added. In addition IPTG was added to one culture to induce <i>mazE</i> expression. The two cultures were compared via the level of protein synthesis and OD600.</br>Finally Amitai et al. confirmed the assumption that the overproduction of MazE after until 6h under overproduction of MazF could resuscitate E.coli cells in LB medium (Fig. 1A)<sup><a href = #671>67.1</a></sup>, but the longer MazF was induced the less cells could be resuscitated by MazE.</br>Whereas MazE overproduction can reverse the inhibitory effect of MazF on translation, it cannot reverse the effect of MazF on colony formation, which is shown in figure 2. Only 1h after the induction of MazE expression, the rate of translation was restored to nearly 100% (Fig.2 Aa, Ab, Ac) but the bacteriocidic effect could not be reversed (Fig.2 Ba, Bb, Bc).</br>Additionally, Amtai et al. found out, that in M9 medium MazE was less able to reverse the effects of MazF overexpression than in LB medium (Fig.1B vs. 1A). it was concluded that there is a point of no return, when MazE is inable to resuscitate a MazF damaged cell, which occurs earlier in M9 medium than in LB medium.</br>Based on their results a model of the MazEF mechanism was built: A <i>mazF</i>-mediated cascade leads to a cell death pathway, but can nevertheless be stopped at several intermediary steps by e.g. <i>mazE</i>. When a point of no return is reached, the cascade cannot be stopped anymore.<div class='content-image' align='center' height=827 width=784><a href='https://static.igem.org/mediawiki/2013/9/9e/Team_Bonn_MazF_2.png'><img src='https://static.igem.org/mediawiki/2013/9/9e/Team_Bonn_MazF_2.png' height=817 width=764></a></br><i>Effect of MazE overproduction during growth in liquid medium on the ability of MazF-overproducing E. coli cells to synthesize proteins and form colonies. To induce <i>mazE</i> expression, IPTG was added to the bacterial culture at 1 h (Aa and Ba), 4 h (Ab and Bb), and 6 h (Ac and Bc) after <i>mazF</i> induction at time zero. The effects of the ectopic overexpression of MazE were measured at 1 and 3 h after the induction of <i>mazE</i> expression.<sup><a href = #671>67.1</a></sup>.</i></div></br>Back to our project and to the idea of a light inducible kill-switch system:</br>As we described in the previous paragraph for both of our kill-switch systems using the MazEF module either MazF or MazE could be degraded:<ul><li>Using the degradation of MazF:</b> For that purpose the insertion of a plasmid containing the ssrA-tagged toxin encoding gene is needed. Since the predominance of the toxin activates a cell death pathway in bacteria, a bacterium containing a module that allows light inducible degradation of the MazF would only be viable, when it is degraded. In darkness the MazF overexpression is no longer compensated and aggregation of it leads to cell death. </li><li><b>Using the degradation of the antitoxin:</b> Two plasmids are needed: The first one to express the MazF and the second one to express the ssra-tagged MazE, in such manner that the amounts of the <i>MazF</i> and the <i>MazE</i> are in equilibrium. Once light induces the degradation system, the MazE is degraded and the predominant toxin will kill the bacterium.</li></ul>As we explained in the general kill-switch system text we finally focused on the second system (via the degradation of MazE).</br>With the design of a MazEF kill-switch system the possibility of resuscitating bacteria in the way Amitai et al. showed has to be considered. A predominant MazF could kill a bacterium in LB within about two hours, but it needs to be predominant over a long period (>7h) to induce its death without it being resuscitated by renewed <i>mazE</i> expression (Fig.1A)with more than 50% probability .</br>Certainly these facts seem to be unfavourable for the realization of a kill switch system via MazEF, but fortunately our system of heterodimerization (Lungu et al.) allows long continuous degradation<sup><a href = #673>67.3</a></sup>, due to the high stability of the light induced hererodimer. Therefore likely a short exposure time will result in prolonged protein degradation sufficing for bacterial death. Additionally, Amitai et al. showed that the less nutrition is available for a bacterium, the earlier the point of no return is reached. If a bacterium escapes the lab, it will likely have less nutrition available than in LB medium. It might reach the point of no return earlier.</br>We described a light inducible MazEF kill-switch system via the insertion of plasmids into bacteria. However a final kill-switch system would have to be implemented in the genomic DNA since plasmids in bacteria can be ejected, for instance via cell division, whereas a genomic DNA mutation is less likely to occur. Nevertheless risk of a loss of function cannot be eliminated , which is why a secure system should countain much more than one kill-switch system to compensate the malfunction of a single kill-switch system. Therefore, we consider the MazEF kill-switch system to be part of a much larger security system for genetically engineered bacteria.<h2>References</h2><a name =671>67.1</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC532418/'>MazF-Mediated Cell Death in Escherichia coli: a Point of No Return, Shahar Amitai et al., Journal of Bacteriology Vol. 186, No. 24, 2004, p.8295–8300.</a></br><a name =672>67.2</a> <a href = 'http://www.ncbi.nlm.nih.gov/pubmed/?term=Rapid+induction+and+reversal+of+bacteriostatic+conditions+by+controlled+expression+of+toxins+and+antitoxins'>Rapid induction and reversal of bacteriostatic conditions by controlled expression of toxins and antitoxins, Pedersen et al., Molecular Microbiology 45, 2002, 501–510.</a></br><a name =673>67.3</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334866/'>Designing Photoswitchable Peptides Using the AsLOV2 Domain, Oana I. Lungu et al., Chem Biol. 2012, 19(4):507-17.</a></br><a name =674>67.4</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC39188/'>An Escherichia coli chromosomal addiction module regulated by guanosine 3,5-bispyrophosphate: a model for programmed bacterial cell death, Aizenman, E., H. Engelberg-Kulka, and G. Glaser, Proc. Natl. Acad. Sci., 1996, USA 93:6059-6063.</a></br><a name =675>67.5</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC28068/'>rexB of bacteriophage lambda is an anti-cell death gene. Engelberg-Kulka, H., M. Reches, S. Narasimhan, R. Schoulaker-Schwarz, Y. Klemes, E. Aizenman, and G. Glaser, Proc. Natl. Acad. Sci., 1998, USA 95:15481-15486.</a></br><a name =676>67.6</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC95100/'>Programmed cell death in Escherichia coli: some antibiotics can trigger mazEF lethality, Sat, B., R. Hazan, T. Fisher, H. Khaner, G. Glaser, and H. Engelberg-Kulka, J. Bacteriol. 2001, 183:2041-2045.</a></br><a name =677>67.7</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC95101/'>Postsegregational killing mediated by the P1 phage addiction module Phd-Doc requires the Escherichia coli programmed cell death system mazEF, Hazan, R., B. Sat, M. Reches, and H. Engelberg-Kulka, J. Bacteriol. 2001, 183:2046–2050.</a></br><a name =678>67.8</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC150121/'>The Escherichia coli mazEF suicide module mediates thymineless death, Sat, B., M. Reches, and H. Engelberg-Kulka, J. Bacteriol. 2003, 185:1803–1807.</a></br><a name =679>67.9</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC415763/'>Escherichia coli mazEFmediated cell death is triggered by various stressful conditions, Hazan, R., B. Sat, and H. Engelberg-Kulka, J. Bacteriol. 2004, 186:3663–3669.</a>"; | content.text= "Our system of light inducible protein degradation can be utilized to degrade any specific protein and is therefore usable to realize a light inducible kill-switch system. A connection of protein degradation to a cell death pathway is represented by the stress-induced toxin-antitoxin module <i>mazEF</i> in Escherichia coli. <i>mazEF</i> is located on the chromosome in E.coli which is associated with programmed cell death. The toxin-antitoxin system is composed by an upstream gene <i>mazE</i>, encoding a labile antitoxin, and a downstream gene mazF, that encodes a stable toxin. </br>The product of <i>mazF</i> cleaves mRNAs and tmRNAs at a specific site, which leads to an inhibition of translation. MazF shows a specific cleaving mechanism, which is not well understood yet, but shows that there is also protein synthesis which is unaffected by MazF. These proteins are presumably part of a cell death pathway. </br>The effect of <i>mazF</i> is suppressed by the Product of <i>mazE</i> which is degraded by the Protease ClpAP in bacteria. As a result of stressful conditions expression of the chromosomal <i>mazEF</i> module is reduced which leads to an imbalance between the products of <i>mazF</i> and <i>mazE</i>: When expression is lowered the stable toxin of <i>mazF</i> still persists while the labile antitoxin of <i>mazE</i> is degraded and can no longer suppress the effect of <i>mazF</i> leading to acute toxicity and cell death. </br><i>MazEF</i>-mediated cell death in E. coli can be caused by:<ul><li>extreme amino acid starvation<sup><a href = #674>67.4</a></sup><sup><a href = #675>67.5</a></sup></li><li> inhibition of transcription and/or translation by antibiotics such as rifampin, chloramphenicol, and spectinomycin under specific growth conditions<sup><a href = #676>67.6</a></sup></li><li>inhibition of translation by the Doc protein of prophage P1<sup><a href = #677>67.7</a></sup></li><li>DNA damage caused by thymine starvation<sup><a href = #678>67.8</a></sup> as well as by mitomycin C, nalidixic acid, and UV irradiation<sup><a href = #679>67.9</a></sup></li><li>oxidative stress (H2O2)<sup><a href = #679'>67.9</a></sup></li></ul>Amitai et al. tested in 2004 the Hypothesis of Pedersen et al.<sup><a href = #672>67.2</a></sup>, that chromosomal toxin-antitoxin systems may rather cause a state of reversible bacteriostasis than programmed cell death<sup><a href = #671>67.1</a></sup>.Therefore E.coli strain MC4100 Δ<i>mazE</i>F relA1 lacIq was cotransformated with:<ul><li>pBad-<i>mazF</i></li><li>pQE-Δhis-<i>mazE</i></li></ul><i>mazF</i>-expression can be induced by the addition of Arabinose via the pBad promoter of the first plasmid. The transformation of the second plasmid results firstly in the repression of <i>mazE</i> expression, whereas when IPTG is added <i>mazE</i> production is induced.</br><div class='content-image' align='center' height=501 width=410><a href='https://static.igem.org/mediawiki/2013/a/ac/Team_Bonn_MazF_1.png'><img src='https://static.igem.org/mediawiki/2013/a/ac/Team_Bonn_MazF_1.png' height=491 width=400></a></br><i>Ability of E. coli cells that had been ectopically overexpressing MazF in liquid medium to form colonies when ectopically overexpressing MazE on plates. The cultures were grown in LB medium (A) or M9 minimal medium with 0.5% glycerol (B) at 37°C to midlogarithmic phase (OD600, 0.5)<sup><a href = #671>67.1</a></sup>.</i></div>Using these tools, Amitai et al. tested the effect of MazE overproduction on MazF-overproducing bacteria during growth in liquid medium.</br>The E.coli strain was incubated in LB medium. After <i>mazF</i> expression was induced by adding arabinose two samples were taken at several time points. To repress <i>mazF</i> expression to both of them glucose was added. In addition IPTG was added to one culture to induce <i>mazE</i> expression. The two cultures were compared via the level of protein synthesis and OD600.</br>Finally Amitai et al. confirmed the assumption that the overproduction of MazE after until 6h under overproduction of MazF could resuscitate E.coli cells in LB medium (Fig. 1A)<sup><a href = #671>67.1</a></sup>, but the longer MazF was induced the less cells could be resuscitated by MazE.</br>Whereas MazE overproduction can reverse the inhibitory effect of MazF on translation, it cannot reverse the effect of MazF on colony formation, which is shown in figure 2. Only 1h after the induction of MazE expression, the rate of translation was restored to nearly 100% (Fig.2 Aa, Ab, Ac) but the bacteriocidic effect could not be reversed (Fig.2 Ba, Bb, Bc).</br>Additionally, Amtai et al. found out, that in M9 medium MazE was less able to reverse the effects of MazF overexpression than in LB medium (Fig.1B vs. 1A). it was concluded that there is a point of no return, when MazE is inable to resuscitate a MazF damaged cell, which occurs earlier in M9 medium than in LB medium.</br>Based on their results a model of the MazEF mechanism was built: A <i>mazF</i>-mediated cascade leads to a cell death pathway, but can nevertheless be stopped at several intermediary steps by e.g. <i>mazE</i>. When a point of no return is reached, the cascade cannot be stopped anymore.<div class='content-image' align='center' height=827 width=784><a href='https://static.igem.org/mediawiki/2013/9/9e/Team_Bonn_MazF_2.png'><img src='https://static.igem.org/mediawiki/2013/9/9e/Team_Bonn_MazF_2.png' height=817 width=764></a></br><i>Effect of MazE overproduction during growth in liquid medium on the ability of MazF-overproducing E. coli cells to synthesize proteins and form colonies. To induce <i>mazE</i> expression, IPTG was added to the bacterial culture at 1 h (Aa and Ba), 4 h (Ab and Bb), and 6 h (Ac and Bc) after <i>mazF</i> induction at time zero. The effects of the ectopic overexpression of MazE were measured at 1 and 3 h after the induction of <i>mazE</i> expression.<sup><a href = #671>67.1</a></sup>.</i></div></br>Back to our project and to the idea of a light inducible kill-switch system:</br>As we described in the previous paragraph for both of our kill-switch systems using the MazEF module either MazF or MazE could be degraded:<ul><li>Using the degradation of MazF:</b> For that purpose the insertion of a plasmid containing the ssrA-tagged toxin encoding gene is needed. Since the predominance of the toxin activates a cell death pathway in bacteria, a bacterium containing a module that allows light inducible degradation of the MazF would only be viable, when it is degraded. In darkness the MazF overexpression is no longer compensated and aggregation of it leads to cell death. </li><li><b>Using the degradation of the antitoxin:</b> Two plasmids are needed: The first one to express the MazF and the second one to express the ssra-tagged MazE, in such manner that the amounts of the <i>MazF</i> and the <i>MazE</i> are in equilibrium. Once light induces the degradation system, the MazE is degraded and the predominant toxin will kill the bacterium.</li></ul>As we explained in the general kill-switch system text we finally focused on the second system (via the degradation of MazE).</br>With the design of a MazEF kill-switch system the possibility of resuscitating bacteria in the way Amitai et al. showed has to be considered. A predominant MazF could kill a bacterium in LB within about two hours, but it needs to be predominant over a long period (>7h) to induce its death without it being resuscitated by renewed <i>mazE</i> expression (Fig.1A)with more than 50% probability .</br>Certainly these facts seem to be unfavourable for the realization of a kill switch system via MazEF, but fortunately our system of heterodimerization (Lungu et al.) allows long continuous degradation<sup><a href = #673>67.3</a></sup>, due to the high stability of the light induced hererodimer. Therefore likely a short exposure time will result in prolonged protein degradation sufficing for bacterial death. Additionally, Amitai et al. showed that the less nutrition is available for a bacterium, the earlier the point of no return is reached. If a bacterium escapes the lab, it will likely have less nutrition available than in LB medium. It might reach the point of no return earlier.</br>We described a light inducible MazEF kill-switch system via the insertion of plasmids into bacteria. However a final kill-switch system would have to be implemented in the genomic DNA since plasmids in bacteria can be ejected, for instance via cell division, whereas a genomic DNA mutation is less likely to occur. Nevertheless risk of a loss of function cannot be eliminated , which is why a secure system should countain much more than one kill-switch system to compensate the malfunction of a single kill-switch system. Therefore, we consider the MazEF kill-switch system to be part of a much larger security system for genetically engineered bacteria.<h2>References</h2><a name =671>67.1</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC532418/'>MazF-Mediated Cell Death in Escherichia coli: a Point of No Return, Shahar Amitai et al., Journal of Bacteriology Vol. 186, No. 24, 2004, p.8295–8300.</a></br><a name =672>67.2</a> <a href = 'http://www.ncbi.nlm.nih.gov/pubmed/?term=Rapid+induction+and+reversal+of+bacteriostatic+conditions+by+controlled+expression+of+toxins+and+antitoxins'>Rapid induction and reversal of bacteriostatic conditions by controlled expression of toxins and antitoxins, Pedersen et al., Molecular Microbiology 45, 2002, 501–510.</a></br><a name =673>67.3</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334866/'>Designing Photoswitchable Peptides Using the AsLOV2 Domain, Oana I. Lungu et al., Chem Biol. 2012, 19(4):507-17.</a></br><a name =674>67.4</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC39188/'>An Escherichia coli chromosomal addiction module regulated by guanosine 3,5-bispyrophosphate: a model for programmed bacterial cell death, Aizenman, E., H. Engelberg-Kulka, and G. Glaser, Proc. Natl. Acad. Sci., 1996, USA 93:6059-6063.</a></br><a name =675>67.5</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC28068/'>rexB of bacteriophage lambda is an anti-cell death gene. Engelberg-Kulka, H., M. Reches, S. Narasimhan, R. Schoulaker-Schwarz, Y. Klemes, E. Aizenman, and G. Glaser, Proc. Natl. Acad. Sci., 1998, USA 95:15481-15486.</a></br><a name =676>67.6</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC95100/'>Programmed cell death in Escherichia coli: some antibiotics can trigger mazEF lethality, Sat, B., R. Hazan, T. Fisher, H. Khaner, G. Glaser, and H. Engelberg-Kulka, J. Bacteriol. 2001, 183:2041-2045.</a></br><a name =677>67.7</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC95101/'>Postsegregational killing mediated by the P1 phage addiction module Phd-Doc requires the Escherichia coli programmed cell death system mazEF, Hazan, R., B. Sat, M. Reches, and H. Engelberg-Kulka, J. Bacteriol. 2001, 183:2046–2050.</a></br><a name =678>67.8</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC150121/'>The Escherichia coli mazEF suicide module mediates thymineless death, Sat, B., M. Reches, and H. Engelberg-Kulka, J. Bacteriol. 2003, 185:1803–1807.</a></br><a name =679>67.9</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC415763/'>Escherichia coli mazEFmediated cell death is triggered by various stressful conditions, Hazan, R., B. Sat, and H. Engelberg-Kulka, J. Bacteriol. 2004, 186:3663–3669.</a>"; | ||
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content.parents=[54]; | content.parents=[54]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "ccdAB"; |
content.titleLong = "The ccd toxin-antitoxin system"; | content.titleLong = "The ccd toxin-antitoxin system"; | ||
content.summary= "CcdB leads to double strand breaks in bacterial genomic DNA and thus is a potent toxin leading to cell death. ccdA compromises the toxic effect of ccdB."; | content.summary= "CcdB leads to double strand breaks in bacterial genomic DNA and thus is a potent toxin leading to cell death. ccdA compromises the toxic effect of ccdB."; | ||
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content.titleShort = "Human Practice"; | content.titleShort = "Human Practice"; | ||
content.titleLong = "Human Practice in General"; | content.titleLong = "Human Practice in General"; | ||
| - | content.summary= " | + | content.summary= "For our Human Practice advances our project's design follows the well-known franchise of Star Wars - being inspired by the LOV domain our project name "LOV WARS" was born. The constant references to the Star Wars movies facilitate the access to synthetic biology to a person that is not familiar with the subject. With the most famous icon of Star Wars being the laser sword it offers a great opportunity to present our light induction system in an entertaining way. We put great effort into adapting every aspect of our public presence to Star Wars."; |
content.text= "<p>The major goal in Human Practice is to make synthetic biology easily understandable and interesting for everybody. For that purpose our project's design follows the well-known franchise of Star Wars; our project name 'LOV WARS' was born. The constant references to the Star Wars movies facilitate the access to synthetic biology to a person that is not familiar with the subject. With the most famous icon of Star Wars being the laser sword it offers a great opportunity to present our light induction system in an entertaining way. We put great effort into adapting every aspect of our public presence to Star Wars.</p><p>Therefore we introduced a <a onclick=node(110)>flash game</a> and a <a onclick=node(109)>comic series</a> called LOV Wars in which we explain basic concepts of synthetic biology and our project. The comic and the LOV Wars shooter arouse interest in people that would normally never engage with the field. That way we can spread the possibilities and advantages that modern genetically engineered organisms offer and clear possible misunderstandings or prejudices against the subject.</p>For the same purpose we organized multiple events for general public at which we presented the tools used in synthetic biology and our project; over the last six months we <a onclick=node(108)>visited several High Schools</a> in Bonn and nearby and gave presentations. In addition we had an information booth in our city center informing passersby about the risks and benefits of synthetic biology with the aid of several posters and flyers. A big highlight was a <a onclick=node(106)>Science slam</a> we arranged. In front of a big audience six scientists from different fields presented an interesting aspect of their academic field. The Science Slam was a great success in terms of getting people in touch with science and without exception received very good critiques.<br> At every opportunity we handed out questionnaires in which we evaluated the public's opinion about synthetic biology and whether our human practice events were informative and interesting."; | content.text= "<p>The major goal in Human Practice is to make synthetic biology easily understandable and interesting for everybody. For that purpose our project's design follows the well-known franchise of Star Wars; our project name 'LOV WARS' was born. The constant references to the Star Wars movies facilitate the access to synthetic biology to a person that is not familiar with the subject. With the most famous icon of Star Wars being the laser sword it offers a great opportunity to present our light induction system in an entertaining way. We put great effort into adapting every aspect of our public presence to Star Wars.</p><p>Therefore we introduced a <a onclick=node(110)>flash game</a> and a <a onclick=node(109)>comic series</a> called LOV Wars in which we explain basic concepts of synthetic biology and our project. The comic and the LOV Wars shooter arouse interest in people that would normally never engage with the field. That way we can spread the possibilities and advantages that modern genetically engineered organisms offer and clear possible misunderstandings or prejudices against the subject.</p>For the same purpose we organized multiple events for general public at which we presented the tools used in synthetic biology and our project; over the last six months we <a onclick=node(108)>visited several High Schools</a> in Bonn and nearby and gave presentations. In addition we had an information booth in our city center informing passersby about the risks and benefits of synthetic biology with the aid of several posters and flyers. A big highlight was a <a onclick=node(106)>Science slam</a> we arranged. In front of a big audience six scientists from different fields presented an interesting aspect of their academic field. The Science Slam was a great success in terms of getting people in touch with science and without exception received very good critiques.<br> At every opportunity we handed out questionnaires in which we evaluated the public's opinion about synthetic biology and whether our human practice events were informative and interesting."; | ||
content.type="Human Practice"; | content.type="Human Practice"; | ||
Revision as of 11:55, 27 October 2013
