http://2013.igem.org/wiki/index.php?title=Team:Hong_Kong_CUHK/backgroundVS&feed=atom&action=historyTeam:Hong Kong CUHK/backgroundVS - Revision history2024-03-28T17:20:57ZRevision history for this page on the wikiMediaWiki 1.16.5http://2013.igem.org/wiki/index.php?title=Team:Hong_Kong_CUHK/backgroundVS&diff=361716&oldid=prevJaneytling at 03:57, 29 October 20132013-10-29T03:57:30Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p>Voltage Switch is a response system triggered by a change of potential across the bacterial inner membrane. Components of this system include the transmembrane voltage sensor peptide originated from potassium ion channels, polyproline linker in the inner membrane, glycine serine linker, PDZ domain and PDZ ligand in the periplasm, and glycine serine linker and effectors in the cytosol.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p>Voltage Switch is a response system triggered by a change of potential across the bacterial inner membrane. Components of this system include the transmembrane voltage sensor peptide originated from potassium ion channels, polyproline linker in the inner membrane, glycine serine linker, PDZ domain and PDZ ligand in the periplasm, and glycine serine linker and effectors in the cytosol.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>1. Components of Voltage Switch System</h3><p align="center"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>1. Components of Voltage Switch System</h3><p align="center"></div></td></tr>
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</table>Janeytlinghttp://2013.igem.org/wiki/index.php?title=Team:Hong_Kong_CUHK/backgroundVS&diff=347489&oldid=prevJaneytling at 15:44, 28 October 20132013-10-28T15:44:08Z<p></p>
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</table>Janeytlinghttp://2013.igem.org/wiki/index.php?title=Team:Hong_Kong_CUHK/backgroundVS&diff=347479&oldid=prevJaneytling at 15:43, 28 October 20132013-10-28T15:43:24Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> These effectors can be changed accordingly. In our project, we chose laccase and dioxygenase for BaP degradation. We also planned to test the system with bimolecular fluorescence complementation (BiFC) dimerization. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> These effectors can be changed accordingly. In our project, we chose laccase and dioxygenase for BaP degradation. We also planned to test the system with bimolecular fluorescence complementation (BiFC) dimerization. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To become functional, the PDZ domain and PDZ ligand bind the two protein fragments together, forming a stable dimer.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To become functional, the PDZ domain and PDZ ligand bind the two protein fragments together, forming a stable dimer.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><h3>2. Proposed Mechanism of Switching &ldquo;ON&rdquo; and &ldquo;OFF&rdquo; in the System<h3></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><h3>2. Proposed Mechanism of Switching &ldquo;ON&rdquo; and &ldquo;OFF&rdquo; in the System<<ins class="diffchange diffchange-inline">/</ins>h3></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong>Switching &ldquo;OFF&rdquo;</strong><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><strong>Switching &ldquo;OFF&rdquo;</strong><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> When no external charge is applied, the two effectors are combined, as the repulsion force between the two positively-charged voltage sensor peptide is relatively smaller than the dimer effect. The two voltage sensor peptides are vertically parallel to each other.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> When no external charge is applied, the two effectors are combined, as the repulsion force between the two positively-charged voltage sensor peptide is relatively smaller than the dimer effect. The two voltage sensor peptides are vertically parallel to each other.</p></div></td></tr>
</table>Janeytlinghttp://2013.igem.org/wiki/index.php?title=Team:Hong_Kong_CUHK/backgroundVS&diff=347461&oldid=prevJaneytling at 15:42, 28 October 20132013-10-28T15:42:24Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p>Voltage Switch is a response system triggered by a change of potential across the bacterial inner membrane. Components of this system include the transmembrane voltage sensor peptide originated from potassium ion channels, polyproline linker in the inner membrane, glycine serine linker, PDZ domain and PDZ ligand in the periplasm, and glycine serine linker and effectors in the cytosol.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p>Voltage Switch is a response system triggered by a change of potential across the bacterial inner membrane. Components of this system include the transmembrane voltage sensor peptide originated from potassium ion channels, polyproline linker in the inner membrane, glycine serine linker, PDZ domain and PDZ ligand in the periplasm, and glycine serine linker and effectors in the cytosol.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>1. Components of Voltage Switch System</h3><p align="center"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>1. Components of Voltage Switch System</h3><p align="center"></div></td></tr>
</table>Janeytlinghttp://2013.igem.org/wiki/index.php?title=Team:Hong_Kong_CUHK/backgroundVS&diff=347437&oldid=prevJaneytling at 15:40, 28 October 20132013-10-28T15:40:57Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <del class="diffchange diffchange-inline"><p2></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <p><ins class="diffchange diffchange-inline">Voltage Switch is </ins>a <ins class="diffchange diffchange-inline">response system triggered by a change </ins>of <ins class="diffchange diffchange-inline">potential </ins> <ins class="diffchange diffchange-inline">across </ins>the <ins class="diffchange diffchange-inline">bacterial inner membrane</ins>. <ins class="diffchange diffchange-inline">Components </ins>of <ins class="diffchange diffchange-inline">this system include the transmembrane </ins> <ins class="diffchange diffchange-inline">voltage sensor peptide originated from potassium </ins>ion channels, <ins class="diffchange diffchange-inline">polyproline </ins> <ins class="diffchange diffchange-inline">linker in </ins>the <ins class="diffchange diffchange-inline">inner </ins>membrane, <ins class="diffchange diffchange-inline">glycine serine linker, PDZ domain </ins>and <ins class="diffchange diffchange-inline">PDZ ligand </ins> <ins class="diffchange diffchange-inline">in </ins>the <ins class="diffchange diffchange-inline">periplasm, </ins>and <ins class="diffchange diffchange-inline">glycine serine linker and effectors </ins>in the <ins class="diffchange diffchange-inline">cytosol</ins>.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> <h1>Background</h1></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><<ins class="diffchange diffchange-inline">h3</ins>><ins class="diffchange diffchange-inline">1. Components of Voltage Switch System</ins></<ins class="diffchange diffchange-inline">h3><p align="center"</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><p><del class="diffchange diffchange-inline">The voltage-dependent ion channels family are </del>a <del class="diffchange diffchange-inline"> group </del>of <del class="diffchange diffchange-inline">ion channel proteins found to have permeability to ions dependent on </del> the <del class="diffchange diffchange-inline">voltage in the external environment</del>. <del class="diffchange diffchange-inline">This family </del>of <del class="diffchange diffchange-inline">ion channels are widely </del> <del class="diffchange diffchange-inline">found in neuronal cells, which transmit signal through electricity pulse. It was found that inside the </del>ion channels, <del class="diffchange diffchange-inline">there exist a few repeating short peptides that are responsible for </del> <del class="diffchange diffchange-inline">voltage sensing and the open and close of the channel. Studies have shown that this peptide can migrate across </del>the membrane <del class="diffchange diffchange-inline">in responds to voltage</del>, and <del class="diffchange diffchange-inline">analysis of </del>the <del class="diffchange diffchange-inline">peptide showed numerous positively charged arginine </del>and <del class="diffchange diffchange-inline"> hydrophobic leucine. However, there were not much applications. In this project we will introduce this voltage sensor peptide as our novel switch, thus achieve high speed respond in bacteria either in form of biomolecular fluorescent complementation, or increased reaction rate </del>in the <del class="diffchange diffchange-inline">degradation pathway of carcinogenic substance BaP</del>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins><<ins class="diffchange diffchange-inline">img width="36" height="36" src="file:///C|/Users/Janecube/AppData/Roaming/Adobe/Dreamweaver CS6/zh_TW/OfficeImageTemp/clip_image001.png" alt="1"</ins>><<ins class="diffchange diffchange-inline">img src="https:</ins>/<ins class="diffchange diffchange-inline">/static.igem.org/mediawiki/2013/8/84/Vs1.png" alt="2" width="521" height="270"> <br</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><<del class="diffchange diffchange-inline">h1</del>><del class="diffchange diffchange-inline">Project Description</del></<del class="diffchange diffchange-inline">h1</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins><<ins class="diffchange diffchange-inline">/</ins>p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><<del class="diffchange diffchange-inline">h3</del>><del class="diffchange diffchange-inline">Voltage Switch</del></<del class="diffchange diffchange-inline">h3</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p<ins class="diffchange diffchange-inline">><stong</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><p><del class="diffchange diffchange-inline">Having inspired by the voltage sensor peptide, we decided to make use of it as our novel voltage switch. </del><<del class="diffchange diffchange-inline">/</del>p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins><<ins class="diffchange diffchange-inline">strong</ins>><ins class="diffchange diffchange-inline">Voltage Sensor Peptide<</ins>/<ins class="diffchange diffchange-inline">strong></ins></<ins class="diffchange diffchange-inline">strong</ins>><<ins class="diffchange diffchange-inline">br</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><<del class="diffchange diffchange-inline">h3</del>><del class="diffchange diffchange-inline">PDZ Domain</del>/<del class="diffchange diffchange-inline">Ligand</del></<del class="diffchange diffchange-inline">h3</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> Voltage sensor peptide, originated from </ins>the <ins class="diffchange diffchange-inline">archaea </ins><em><ins class="diffchange diffchange-inline">Aeropyrum pernix</ins></em><ins class="diffchange diffchange-inline">,&nbsp;is </ins>a <ins class="diffchange diffchange-inline">protein domain </ins> <ins class="diffchange diffchange-inline">found in a </ins>voltage<ins class="diffchange diffchange-inline">-dependent potassium ion channel</ins>. <ins class="diffchange diffchange-inline">Previous studies suggested that </ins> the domain <ins class="diffchange diffchange-inline">could translocate within the membrane upon membrane potential change </ins> <ins class="diffchange diffchange-inline">[10, 11]</ins>. <ins class="diffchange diffchange-inline">This helix peptide received its voltage-sensing ability from its four </ins> positively<ins class="diffchange diffchange-inline">-</ins>charged <ins class="diffchange diffchange-inline">arginine residues</ins>, and <ins class="diffchange diffchange-inline">high hydrophobicity from its numerous </ins> <ins class="diffchange diffchange-inline">leucine residues</ins>.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><<del class="diffchange diffchange-inline">p</del>><del class="diffchange diffchange-inline">We made use of </del>the <del class="diffchange diffchange-inline">PDZ domain and ligand found in </del><em><del class="diffchange diffchange-inline">Mus musculus</del></em> <del class="diffchange diffchange-inline">as </del>a <del class="diffchange diffchange-inline">pair of </del> <del class="diffchange diffchange-inline">dimers that link two </del>voltage <del class="diffchange diffchange-inline">sensor peptide together</del>. <del class="diffchange diffchange-inline">As demonstrated by the </del> <del class="diffchange diffchange-inline">SJTU iGEM team of 2012, </del>the <del class="diffchange diffchange-inline">PDZ </del>domain <del class="diffchange diffchange-inline">and ligand functioned and worked as a </del> <del class="diffchange diffchange-inline">membrane protein scaffolding part which links enzymes together through linkers</del>. <del class="diffchange diffchange-inline">Since both voltage sensor peptides are </del>positively charged, <del class="diffchange diffchange-inline">without the help of the PDZ domain </del>and <del class="diffchange diffchange-inline">ligand, they would not bind together due to mutual </del> <del class="diffchange diffchange-inline">repulsion. With the PDZ domain and ligand, the 2 voltage sensor peptides would dimerize, thus forming the complete voltage switch</del>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><<ins class="diffchange diffchange-inline">p</ins>><ins class="diffchange diffchange-inline"><strong>PDZ Domain and PDZ Ligand</ins></<ins class="diffchange diffchange-inline">strong</ins>><<ins class="diffchange diffchange-inline">br</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><<del class="diffchange diffchange-inline">h3</del>><del class="diffchange diffchange-inline">Voltage Sensor Peptide</del></<del class="diffchange diffchange-inline">h3</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">This is a dimer found </ins>in <em><ins class="diffchange diffchange-inline">Mus musculus</ins></em>, <ins class="diffchange diffchange-inline">and were used by </ins>the <ins class="diffchange diffchange-inline">2012 SJTU iGEM team as </ins>a <ins class="diffchange diffchange-inline">membrane </ins> <ins class="diffchange diffchange-inline">scaffolding tool</ins>. The <ins class="diffchange diffchange-inline">PDZ dimer provides </ins>the <ins class="diffchange diffchange-inline">necessary linkage of </ins>the <ins class="diffchange diffchange-inline">two fusion </ins> <ins class="diffchange diffchange-inline">proteins </ins>in the <ins class="diffchange diffchange-inline">voltage switch system</ins>.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><<del class="diffchange diffchange-inline">p</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><<ins class="diffchange diffchange-inline">p</ins>><ins class="diffchange diffchange-inline"><strong>Polyproline Linker</ins></<ins class="diffchange diffchange-inline">strong</ins>><<ins class="diffchange diffchange-inline">br</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> Found </del>in <em><del class="diffchange diffchange-inline">Aeropyrum pernix</del></em>, the <del class="diffchange diffchange-inline">voltage sensor peptide contains arginine residues along the peptide, which give the peptide </del>a <del class="diffchange diffchange-inline">net </del> <del class="diffchange diffchange-inline">positive charge under phyiological pH</del>. <del class="diffchange diffchange-inline">It also contains a lot of leucine and other hydrophobic residues, which make it an excellent transmembrane domain. </del>The <del class="diffchange diffchange-inline">voltage sensor peptide itself is very short. We used a polyproline rigid linker to elongate it so that </del>the <del class="diffchange diffchange-inline">separation distance would be longer. In order to link </del>the <del class="diffchange diffchange-inline">part with the PDZ domain and ligand, and also the downstream </del> <del class="diffchange diffchange-inline">effectors, we added </del>in <del class="diffchange diffchange-inline">a polyglycine linker in front and after </del>the <del class="diffchange diffchange-inline">whole part. The rotatable angles on glycine residues allow flexible movement of the whole part</del>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> Polyproline linker is a pure proline rigid rod. Its rigidity is due to </ins> <ins class="diffchange diffchange-inline">the limited rotation </ins>of <ins class="diffchange diffchange-inline">peptide bonds in proline, a ring-shaped amino acid. Also, proline does not contain any hydrophilic functional group, which makes the linker hydrophobic </ins>and <ins class="diffchange diffchange-inline">thus able </ins>to <ins class="diffchange diffchange-inline">stay within </ins>the <ins class="diffchange diffchange-inline">inner membrane</ins>.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><<del class="diffchange diffchange-inline">h3</del>><del class="diffchange diffchange-inline">Effectors</del></<del class="diffchange diffchange-inline">h3</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><ins class="diffchange diffchange-inline"><strong>Glycine Serine Linker</ins></<ins class="diffchange diffchange-inline">strong</ins>><<ins class="diffchange diffchange-inline">br</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><<del class="diffchange diffchange-inline">p</del>><del class="diffchange diffchange-inline">We </del> <del class="diffchange diffchange-inline">made use </del>of <del class="diffchange diffchange-inline">Biomolecular fluorescent complementation </del>and <del class="diffchange diffchange-inline">2 enzymes </del>to <del class="diffchange diffchange-inline"> demonstrate how </del>the <del class="diffchange diffchange-inline">voltage sensors worked</del>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">This </ins>is a <ins class="diffchange diffchange-inline">hydrophilic flexible linker which links PDZ dimer with voltage </ins> <ins class="diffchange diffchange-inline">sensor peptides</ins>. <ins class="diffchange diffchange-inline">It also links </ins>the <ins class="diffchange diffchange-inline">polyproline linkers </ins>in the <ins class="diffchange diffchange-inline">membrane </ins>with the <ins class="diffchange diffchange-inline">effectors in </ins>the <ins class="diffchange diffchange-inline">cytosol</ins>.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><p><del class="diffchange diffchange-inline">Biomoleular fluorescent complementation (BiFC)</del></<del class="diffchange diffchange-inline">p</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><<ins class="diffchange diffchange-inline">p</ins>><ins class="diffchange diffchange-inline"><strong>Effectors</ins></<ins class="diffchange diffchange-inline">strong</ins>><<ins class="diffchange diffchange-inline">br</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><<del class="diffchange diffchange-inline">p</del>><del class="diffchange diffchange-inline">BiFC </del>is a <del class="diffchange diffchange-inline">phenomenon found in fluorescent proteins that, if we cleaved the protein </del> <del class="diffchange diffchange-inline">at some specific sites in the loops, we can separate the protein into 2 parts, the N-terminal and the C-terminal</del>. <del class="diffchange diffchange-inline">These two parts are non-fluorescence, and under normal physiological condition, they will not refold with </del>the <del class="diffchange diffchange-inline">other parts to give fluorescent. However, when there is protein-protein interactions </del>in <del class="diffchange diffchange-inline"> which </del>the <del class="diffchange diffchange-inline">interacting proteins are linking </del>with the <del class="diffchange diffchange-inline">fragments, the interactions </del> <del class="diffchange diffchange-inline">can bring </del>the <del class="diffchange diffchange-inline">two fragments together, where they refold and mature to give fluorescent again. We make use of this feature to be our reporter. When the switch is OFF, the fragments were far apart, so they don&rsquo;t fluoresce, but when we turn ON the switch, it brings the fragments together, thus the fragments refold to give fluoresence. One drawback of this system is that there were still no reported reversible BiFC reaction, and so theoretically it can&rsquo;t be switched off</del>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> These effectors </ins>can be <ins class="diffchange diffchange-inline">changed accordingly</ins>. <ins class="diffchange diffchange-inline">In our project</ins>, <ins class="diffchange diffchange-inline">we chose </ins> <ins class="diffchange diffchange-inline">laccase </ins>and <ins class="diffchange diffchange-inline">dioxygenase for BaP degradation</ins>. We <ins class="diffchange diffchange-inline">also planned </ins>to <ins class="diffchange diffchange-inline">test the system </ins> <ins class="diffchange diffchange-inline">with bimolecular fluorescence complementation (BiFC) dimerization. </ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><<del class="diffchange diffchange-inline">h3</del>><del class="diffchange diffchange-inline">BaP Degradation</del></<del class="diffchange diffchange-inline">h3</del>><<del class="diffchange diffchange-inline">p</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">To become functional, </ins>the <ins class="diffchange diffchange-inline">PDZ domain and PDZ ligand bind </ins>the <ins class="diffchange diffchange-inline">two protein </ins> <ins class="diffchange diffchange-inline">fragments together</ins>, <ins class="diffchange diffchange-inline">forming a stable dimer</ins>.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> Laccase and Dioxygenase are 2 enzymes found in different organisms, which </del>can <del class="diffchange diffchange-inline"> participate in a pathway in which carcinogenic substances BaP is first degraded into quinone, and then to some simple carboxylic acids that could either </del>be <del class="diffchange diffchange-inline"> harmless or very little harmful</del>. <del class="diffchange diffchange-inline">Normally</del>, <del class="diffchange diffchange-inline">the enzymatic reaction rate in cells </del> <del class="diffchange diffchange-inline">won&rsquo;t be very high regardless of the enzyme activity, </del>and <del class="diffchange diffchange-inline">this is because in physiological conditions and most experimental setups, rate of diffusion of substrates is the major limiting factor</del>. We <del class="diffchange diffchange-inline">try </del>to <del class="diffchange diffchange-inline">make use of our voltage </del> <del class="diffchange diffchange-inline">switch as a way to quickly alter </del>the <del class="diffchange diffchange-inline">distance between enzymes to change </del>the <del class="diffchange diffchange-inline">substrate diffusion distance</del>, <del class="diffchange diffchange-inline">thus the reaction rate can be controlled by electricity</del>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p></ins><h3><ins class="diffchange diffchange-inline">2. Proposed Mechanism of Switching &ldquo;ON&rdquo; and &ldquo;OFF&rdquo; in the System</ins><h3<ins class="diffchange diffchange-inline">></p</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><h3><del class="diffchange diffchange-inline">How It Works</del><<del class="diffchange diffchange-inline">/</del>h3></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><<ins class="diffchange diffchange-inline">strong</ins>><ins class="diffchange diffchange-inline">Switching &ldquo;OFF&rdquo;</ins></<ins class="diffchange diffchange-inline">strong</ins>><ins class="diffchange diffchange-inline"><br></ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><p><del class="diffchange diffchange-inline">The voltage switch is a novel protein switch that responds to external voltage. The switch itself consist of the PDZ Ligand-Voltage sensor peptide (</del><<del class="diffchange diffchange-inline">a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1092007"</del>><del class="diffchange diffchange-inline">BBa_K1092007</del></<del class="diffchange diffchange-inline">a</del>><del class="diffchange diffchange-inline">) and </del> the <del class="diffchange diffchange-inline">PDZ Domain</del>-<del class="diffchange diffchange-inline">Voltage </del>sensor peptide <del class="diffchange diffchange-inline">(<a href="http://parts</del>.<del class="diffchange diffchange-inline">igem</del>.<del class="diffchange diffchange-inline">org/wiki/index.php?title=Part:BBa_K1092008">BBa_K1092008</del></<del class="diffchange diffchange-inline">a</del>><del class="diffchange diffchange-inline">), and can be linked to different effectors such as the Dioxygenase (</del><<del class="diffchange diffchange-inline">a href</del>="<del class="diffchange diffchange-inline">http://parts.igem.org/wiki/index.php?title=Part:BBa_K1092002</del>"><del class="diffchange diffchange-inline">BBa_K1092002</del><<del class="diffchange diffchange-inline">/a>) and Laccase (<a href</del>="<del class="diffchange diffchange-inline">http</del>://<del class="diffchange diffchange-inline">parts</del>.igem.org/<del class="diffchange diffchange-inline">wiki</del>/<del class="diffchange diffchange-inline">index</del>.<del class="diffchange diffchange-inline">php?title</del>=<del class="diffchange diffchange-inline">Part:BBa_K1092004</del>"<del class="diffchange diffchange-inline">>BBa_K1092004</a>), or the RFP fragments (<a href</del>="<del class="diffchange diffchange-inline">http://parts.igem.org/wiki/index.php?title</del>=<del class="diffchange diffchange-inline">Part:BBa_K1092105</del>"><del class="diffchange diffchange-inline">BBa_K1092105</del></<del class="diffchange diffchange-inline">a</del>> &<del class="diffchange diffchange-inline">amp</del>; <<del class="diffchange diffchange-inline">a href="http:</del>/<del class="diffchange diffchange-inline">/parts.igem.org/wiki/index.php?title=Part:BBa_K1092106"</del>><del class="diffchange diffchange-inline">BBa_K1092106</del><<del class="diffchange diffchange-inline">/a</del>><del class="diffchange diffchange-inline">). Initially</del>, the two <del class="diffchange diffchange-inline">proteins would express and localize onto the inner membrane </del> <del class="diffchange diffchange-inline">of the bacteria</del>. <del class="diffchange diffchange-inline">The two peptides would then come together forming a dimer. After that, due to </del>the <del class="diffchange diffchange-inline">mutual repulsion of </del>the <del class="diffchange diffchange-inline">positive charges in the two </del> voltage sensor peptide, <del class="diffchange diffchange-inline">the two </del>voltage sensor peptide <del class="diffchange diffchange-inline">would separate (Fig. 1)</del>. <del class="diffchange diffchange-inline">This separate the two effectors down below the two peptide</del>, <del class="diffchange diffchange-inline">causing either a long distance for the </del>two <del class="diffchange diffchange-inline">effectors to interact, or a longer distance of diffusion of substrates. This represent the OFF stage of the voltage switch, and it could be enhanced by using negatively charged environment to further </del>pull <del class="diffchange diffchange-inline">them apart.<br /></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> When no external charge is applied, the two effectors are combined, as </ins> the <ins class="diffchange diffchange-inline">repulsion force between the two positively</ins>-<ins class="diffchange diffchange-inline">charged voltage </ins>sensor peptide <ins class="diffchange diffchange-inline"> is relatively smaller than the dimer effect</ins>. <ins class="diffchange diffchange-inline">The two voltage sensor peptides are vertically parallel to each other</ins>.</<ins class="diffchange diffchange-inline">p</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> <br /></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><<ins class="diffchange diffchange-inline">p align</ins>="<ins class="diffchange diffchange-inline">center</ins>"><<ins class="diffchange diffchange-inline">img src</ins>="<ins class="diffchange diffchange-inline">https</ins>://<ins class="diffchange diffchange-inline">static</ins>.igem.org/<ins class="diffchange diffchange-inline">mediawiki/2013</ins>/<ins class="diffchange diffchange-inline">0/0f/Vs2</ins>.<ins class="diffchange diffchange-inline">png" alt</ins>="<ins class="diffchange diffchange-inline">3" width</ins>="<ins class="diffchange diffchange-inline">472" height</ins>=<ins class="diffchange diffchange-inline">"288</ins>"></<ins class="diffchange diffchange-inline">p</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> During the ON stage, the environment become positively charged, and this environment exert a strong electrostatic repulsion on </del>the two voltage sensor <del class="diffchange diffchange-inline"> peptide so that they can be pushed together by overcoming the mutual repulsion between the </del>peptides <del class="diffchange diffchange-inline">(Fig. 2). This action brings the two downstream effectors </del> <del class="diffchange diffchange-inline">together</del>, which <del class="diffchange diffchange-inline">either provides short-enough distance for them to interact, or greatly reduces </del>the <del class="diffchange diffchange-inline">distance of diffusion </del>of the <del class="diffchange diffchange-inline">substrates, thus greatly enhance the reaction rate</del>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p><strong>Switching </ins>&<ins class="diffchange diffchange-inline">ldquo;ON&rdquo</ins>;</<ins class="diffchange diffchange-inline">strong</ins>><<ins class="diffchange diffchange-inline">br</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><del class="diffchange diffchange-inline">&nbsp;</del></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> When negative charge is applied into the system</ins>, the two <ins class="diffchange diffchange-inline">effectors are </ins> <ins class="diffchange diffchange-inline">separated</ins>. <ins class="diffchange diffchange-inline">As </ins>the <ins class="diffchange diffchange-inline">potential across </ins>the <ins class="diffchange diffchange-inline">membrane is inverted, there is an attractive force between negatively-charged periplasm and positively-charged </ins> voltage sensor peptide, <ins class="diffchange diffchange-inline">and vice versa for cytosol and </ins>voltage sensor peptide. <ins class="diffchange diffchange-inline">Together</ins>, <ins class="diffchange diffchange-inline">these </ins>two <ins class="diffchange diffchange-inline">forces </ins>pull the two voltage sensor peptides <ins class="diffchange diffchange-inline">from vertical </ins> <ins class="diffchange diffchange-inline">position to more horizontal</ins>, which <ins class="diffchange diffchange-inline">causes </ins>the <ins class="diffchange diffchange-inline">separation </ins>of the <ins class="diffchange diffchange-inline">effectors</ins>.</p></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p <ins class="diffchange diffchange-inline">align="center"</ins>><ins class="diffchange diffchange-inline"><img src="https://static.igem.org/mediawiki/2013/d/d3/Vs3.png" alt="4" width="482" height="292"><br clear="all"></ins></div></td></tr>
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</table>Janeytlinghttp://2013.igem.org/wiki/index.php?title=Team:Hong_Kong_CUHK/backgroundVS&diff=346348&oldid=prevChristar at 14:11, 28 October 20132013-10-28T14:11:50Z<p></p>
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</table>Christarhttp://2013.igem.org/wiki/index.php?title=Team:Hong_Kong_CUHK/backgroundVS&diff=335667&oldid=prevJaneytling at 09:02, 27 October 20132013-10-27T09:02:57Z<p></p>
<a href="http://2013.igem.org/wiki/index.php?title=Team:Hong_Kong_CUHK/backgroundVS&diff=335667&oldid=324022">Show changes</a>Janeytlinghttp://2013.igem.org/wiki/index.php?title=Team:Hong_Kong_CUHK/backgroundVS&diff=324022&oldid=prevShengry0716 at 06:46, 22 October 20132013-10-22T06:46:49Z<p></p>
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</table>Shengry0716http://2013.igem.org/wiki/index.php?title=Team:Hong_Kong_CUHK/backgroundVS&diff=323763&oldid=prevShengry0716 at 01:53, 22 October 20132013-10-22T01:53:24Z<p></p>
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<h2>Voltage Switch</h2><br />
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<h1>Background</h1><br />
<p>The voltage-dependent ion channels family are a group of ion channel proteins found to have permeability to ions dependent on the voltage in the external environment. This family of ion channels are widely found in neuronal cells, which transmit signal through electricity pulse. It was found that inside the ion channels, there exist a few repeating short peptides that are responsible for voltage sensing and the open and close of the channel. Studies have shown that this peptide can migrate across the membrane in responds to voltage, and analysis of the peptide showed numerous positively charged arginine and hydrophobic leucine. However, there were not much applications. In this project we will introduce this voltage sensor peptide as our novel switch, thus achieve high speed respond in bacteria either in form of biomolecular fluorescent complementation, or increased reaction rate in the degradation pathway of carcinogenic substance BaP.</p><br />
<h1>Project Description</h1><br />
<h3>~Voltage Switch</h3><br />
<p>Having inspired by the voltage sensor peptide, we decided to make use of it as our novel voltage switch. </p><br />
<h3>~PDZ Domain/Ligand</h3><br />
<p>We made use of the PDZ domain and ligand found in <em>Mus musculus</em> as a pair of dimers that link two voltage sensor peptide together. As demonstrated by the SJTU iGEM team of 2012, the PDZ domain and ligand functioned and worked as a membrane protein scaffolding part which links enzymes together through linkers. Since both voltage sensor peptides are positively charged, without the help of the PDZ domain and ligand, they would not bind together due to mutual repulsion. With the PDZ domain and ligand, the 2 voltage sensor peptides would dimerize, thus forming the complete voltage switch.</p><br />
<h3>~Voltage Sensor Peptide</h3><br />
<p><br />
Found in <em>Aeropyrum pernix</em>, the voltage sensor peptide contains arginine residues along the peptide, which give the peptide a net positive charge under phyiological pH. It also contains a lot of leucine and other hydrophobic residues, which make it an excellent transmembrane domain. The voltage sensor peptide itself is very short. We used a polyproline rigid linker to elongate it so that the separation distance would be longer. In order to link the part with the PDZ domain and ligand, and also the downstream effectors, we added in a polyglycine linker in front and after the whole part. The rotatable angles on glycine residues allow flexible movement of the whole part.</p><br />
<h3>~Effectors</h3><br />
<p>We made use of Biomolecular fluorescent complementation and 2 enzymes to demonstrate how the voltage sensors worked.</p><br />
<p>Biomoleular fluorescent complementation (BiFC)</p><br />
<p>BiFC is a phenomenon found in fluorescent proteins that, if we cleaved the protein at some specific sites in the loops, we can separate the protein into 2 parts, the N-terminal and the C-terminal. These two parts are non-fluorescence, and under normal physiological condition, they will not refold with the other parts to give fluorescent. However, when there is protein-protein interactions in which the interacting proteins are linking with the fragments, the interactions can bring the two fragments together, where they refold and mature to give fluorescent again. We make use of this feature to be our reporter. When the switch is OFF, the fragments were far apart, so they don&rsquo;t fluoresce, but when we turn ON the switch, it brings the fragments together, thus the fragments refold to give fluoresence. One drawback of this system is that there were still no reported reversible BiFC reaction, and so theoretically it can&rsquo;t be switched off.</p><br />
<h3>~BaP Degradation</h3><p><br />
Laccase and Dioxygenase are 2 enzymes found in different organisms, which can participate in a pathway in which carcinogenic substances BaP is first degraded into quinone, and then to some simple carboxylic acids that could either be harmless or very little harmful. Normally, the enzymatic reaction rate in cells won&rsquo;t be very high regardless of the enzyme activity, and this is because in physiological conditions and most experimental setups, rate of diffusion of substrates is the major limiting factor. We try to make use of our voltage switch as a way to quickly alter the distance between enzymes to change the substrate diffusion distance, thus the reaction rate can be controlled by electricity.</p><br />
<h3>~How It Works</h3><br />
<p>The voltage switch is a novel protein switch that responds to external voltage. The switch itself consist of the PDZ Ligand-Voltage sensor peptide (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1092007">BBa_K1092007</a>) and the PDZ Domain-Voltage sensor peptide (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1092008">BBa_K1092008</a>), and can be linked to different effectors such as the Dioxygenase (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1092002">BBa_K1092002</a>) and Laccase (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1092004">BBa_K1092004</a>), or the RFP fragments (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1092105">BBa_K1092105</a> &amp; <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1092106">BBa_K1092106</a>). Initially, the two proteins would express and localize onto the inner membrane of the bacteria. The two peptides would then come together forming a dimer. After that, due to the mutual repulsion of the positive charges in the two voltage sensor peptide, the two voltage sensor peptide would separate (Fig. 1). This separate the two effectors down below the two peptide, causing either a long distance for the two effectors to interact, or a longer distance of diffusion of substrates. This represent the OFF stage of the voltage switch, and it could be enhanced by using negatively charged environment to further pull them apart.<br /><br />
<br /><br />
During the ON stage, the environment become positively charged, and this environment exert a strong electrostatic repulsion on the two voltage sensor peptide so that they can be pushed together by overcoming the mutual repulsion between the peptides (Fig. 2). This action brings the two downstream effectors together, which either provides short-enough distance for them to interact, or greatly reduces the distance of diffusion of the substrates, thus greatly enhance the reaction rate.</p><br />
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