Biomod/2011/Slovenia/BioNanoWizards/resultsproteintethers

From OpenWetWare
Jump to: navigation, search

<html> <head>

 <style>
  1. globalWrapper {width: 1000px; padding:0; border:0; margin:0 auto 0 auto;}

@media screen { body { background: #E3E3E3 0 0 0 0 no-repeat; /* changed default background */ } }

  1. column-one {display:none; width:0px;}
  2. contentSub {display:none; width:0px;}
  3. column-content {border:0px; width: 1000px; padding:0; margin:0;}

.firstHeading {display:none;}

  1. footer {display:none;}
  2. content {background-color: #e3e3e3; border:0; padding:0; margin:0;}
  3. bodyContent {background: none; border:0x; padding0; margin:0;}
  4. header{display:none;}
 </style>
 <style type="text/css">

/* Design by Free CSS Templates http://www.freecsstemplates.org Released for free under a Creative Commons Attribution 2.5 License

  • /

body { margin: 0; padding:0; background: #E3E3E3; font-family: Arial, Helvetica, sans-serif; font-size: 12px; color: #616161; } p, ul, ol { margin-top: 0; line-height: 180%; } ul, ol { } a { text-decoration: none; color: #274775; } a:hover { }

  1. wrapper {

margin: 0 auto; padding: 0; } .container { width: 1000px; margin: 0px auto; } /* Header */

  1. headercss {

width: 900px; height: 150px; margin: 0 auto; padding: 0px 50px; background: url(http://openwetware.org/images/c/cc/Img01.PNG) no-repeat left top; position: relative; z-index:10; } /* Logo */

  1. logo {

width: 800px; margin: 0 0 0 0; padding: 30px 0 0 0; }

  1. logolinknabiomod {

width: 800px; margin: 0 0 0 0; padding: 0 0 0 0; }

  1. logolinknabiomod h1, #logo p {

}

  1. logolinknabiomod h1 {

padding: 60px 0px 0px 0px; letter-spacing: -2px; font-size: 3.8em; background: none; }

  1. logolinknabiomod p {

margin: 0 0 0 0; padding: 0 0 0 0; letter-spacing: -1px; font: normal 14px Georgia, "Times New Roman", Times, serif; font-style: normal; color: #8e8e8e; } #logolinknabiomod p a { color: #8E8E8E; }

  1. logolinknabiomod a {

border: none; background: none; text-decoration: none; } /* Splash */

  1. splash {

height: 300px; } /* Menu */ /* Page */

  1. page {

width: 890px; margin: 0 auto; padding: 30px 55px; background: url(http://openwetware.org/images/e/e6/Lepidimg03.jpg) repeat-y left top; } /* Content */

  1. content {

float: left; width: 890px; padding: 0px 0px 0px 0px; background: none; } .post .entry { text-align: justify; font: Arial; letter-spacing: 0px; z-index:5; } /* Three Column Footer Content */

  1. footer-content {

background: url(http://openwetware.org/images/e/e3/Img04brezcrne.JPG) repeat-y left top; color: #BFBFBF; }

  1. footer-bg {

overflow: hidden; width: 890px; padding: 10px 55px 80px 55px; background: url(http://openwetware.org/images/0/06/Footerbrezozadja.jpg) no-repeat left bottom; }

  1. footer-content h2 {

margin: 0px; padding: 0px 0px 20px 0px; letter-spacing: -1px; text-transform: lowercase; font-size: 26px; color: #202020; }

  1. footer-content ul {

margin: 0px; padding: 0px 0px 0px 0px; }

  1. footer-content a {

color: #447ECF; }

  1. column1 {

float: left; width: 423px; margin: 0px 0px 0px 0px; text-align: justify; }

  1. column2 {

float: right; width: 423px; margin: 0px 0px 0px 0px; text-align: justify; } /* Footer */

  1. footerCSS {

height: 20px; width: 600px; padding: 0px 200px 0px 200px; font-family: Arial, Helvetica, sans-serif; }

  1. footerCSS p {

margin: 0; padding-top: 0px; line-height: normal; font-size: 9px; text-align: center; color: #202020; }

  1. footerCSS a {

color: #202020; }

  1. marketing {

overflow: hidden; margin-bottom: 30px; padding: 20px 0px 10px 0px; border-top: 1px solid #E3E3E3; border-bottom: 1px solid #E3E3E3; }

  1. marketing .text1 {

float: left; margin: 0px; padding: 0 0 0 100px; font-size: 34px; color: #345E9B; }

  1. marketing .text2 {

float: right; }

  1. marketing .text2 a {

display: block; width: 252px; height: 38px; padding: 15px 100px 0px 0px; background: url(http://openwetware.org/images/f/f4/Lepidimg07.jpg) no-repeat left top; letter-spacing: -2px; text-align: center; font-size: 30px; color: #ffffff; }

  1. jebenimeni {

width: 800px; height: 30px; padding: 45px 0 0 0;} <!-- Start PureCSSMenu.com STYLE -->

  1. pcm{display:none;}

ul.pureCssMenu ul{display:none} ul.pureCssMenu li:hover>ul{display:block} ul.pureCssMenu ul{position: absolute;left:-1px;top:98%;} ul.pureCssMenu ul ul{position: absolute;left:98%;top:-2px;} ul.pureCssMenu,ul.pureCssMenu ul { margin:0px; list-style:none; padding:0px 2px 2px 0px; background-color:#2A4B7D; background-repeat:repeat; border-color:#cccccc #111111 #111111 #cccccc; border-width:1px; border-style:solid; } ul.pureCssMenu table {border-collapse:collapse}ul.pureCssMenu { display:block; zoom:1; float: left; } ul.pureCssMenu ul{ width:260.40000000000003px; } ul.pureCssMenu li{ display:block; margin:2px 0px 0px 2px; font-size:0px; } ul.pureCssMenu a:active, ul.pureCssMenu a:focus { outline-style:none; } ul.pureCssMenu a, ul.pureCssMenu li.dis a:hover, ul.pureCssMenu li.sep a:hover { display:block; vertical-align:middle; background-color:#2A4B7D; border-width:1px; border-color:#2A4B7D; border-style:solid; text-align:left; text-decoration:none; padding:2px 5px 2px 10px; _padding-left:0; font:16px Trebuchet MS; color: #ffffff; text-decoration:none; cursor:default; } ul.pureCssMenu span{ overflow:hidden; } ul.pureCssMenu li { float:left; } ul.pureCssMenu ul li { float:none; } ul.pureCssMenu ul a { text-align:left; white-space:nowrap; } ul.pureCssMenu li.sep{ text-align:left; padding:0px; line-height:0; height:100%; } ul.pureCssMenu li.sep span{ float:none; padding-right:0; width:3px; height:100%; display:inline-block; background-color:#cccccc #111111 #111111 #cccccc; background-image:none;} ul.pureCssMenu ul li.sep span{ width:100%; height:3px; } ul.pureCssMenu li:hover{ position:relative; } ul.pureCssMenu li:hover>a{ background-color:#377D9F; border-color:#377D9F; border-style:solid; font:16px Trebuchet MS; color: #FFFFFF; text-decoration:none; } ul.pureCssMenu li a:hover{ position:relative; background-color:#377D9F; border-color:#377D9F; border-style:solid; font:16px Trebuchet MS; color: #FFFFFF; text-decoration:none; } ul.pureCssMenu li.dis a { color: #666 !important; } ul.pureCssMenu img {border: none;float:left;_float:none;margin-right:2px;width:16px; height:16px; } ul.pureCssMenu ul img {width:16px; height:16px; } ul.pureCssMenu img.over{display:none} ul.pureCssMenu li.dis a:hover img.over{display:none !important} ul.pureCssMenu li.dis a:hover img.def {display:inline !important} ul.pureCssMenu li:hover > a img.def {display:none} ul.pureCssMenu li:hover > a img.over {display:inline} ul.pureCssMenu a:hover img.over,ul.pureCssMenu a:hover ul img.def,ul.pureCssMenu a:hover a:hover ul img.def,ul.pureCssMenu a:hover a:hover img.over,ul.pureCssMenu a:hover a:hover a:hover img.over{display:inline} ul.pureCssMenu a:hover img.def,ul.pureCssMenu a:hover ul img.over,ul.pureCssMenu a:hover a:hover ul img.over,ul.pureCssMenu a:hover a:hover img.def,ul.pureCssMenu a:hover a:hover a:hover img.def{display:none} ul.pureCssMenu a:hover ul,ul.pureCssMenu a:hover a:hover ul{display:block} ul.pureCssMenu a:hover ul ul{display:none} ul.pureCssMenu span{ display:block; background-image:url(http://openwetware.org/images/5/50/Arr_white.gif); background-position:right center; background-repeat: no-repeat; padding-right:12px;} ul.pureCssMenu li:hover>a>span{ background-image:url(http://openwetware.org/images/6/6f/Arrv_white.gif); } ul.pureCssMenu a:hover span{ _background-image:url(http://openwetware.org/images/6/6f/Arrv_white.gif)} ul.pureCssMenu ul span,ul.pureCssMenu a:hover table span{background-image:url(http://openwetware.org/images/5/50/Arr_white.gif)} <!-- End PureCSSMenu.com STYLE -->

 </style>

</head> <body> <div id="wrapper"> <div id="headercss" class="container"> <div id="logo"> <div id="logolinknabiomod"> <p><a href="http://openwetware.org/wiki/Biomod/2011">&lt;-- Back to BioMod 2011</a></p> </div> <div id="jebenimeni"> <!-- Start PureCSSMenu.com MENU --> <ul class="pureCssMenu pureCssMenum">

 <li class="pureCssMenui"><a class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards">Home</a></li>
 <li class="pureCssMenui"><a class="pureCssMenui"
href="#"><span>Idea</span></a><!--[if lte IE 6]><table><tr><td><![endif]-->
   <ul class="pureCssMenum">
     <li class="pureCssMenui"><a class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/ideastateoftheart">State

of the art</a></li>

     <li class="pureCssMenui"><a class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/ideaproteinaddons">Protein

add-ons</a></li> <!-- TUKI SEM IZBRISAL PROTEIN CHIMERAS -->

     <li class="pureCssMenui"><a class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/ideaverticalstacks">Vertical

stacks</a></li>

   </ul>

<!--[if lte IE 6]></td></tr></table></a><![endif]--></li>

 <li class="pureCssMenui"><a class="pureCssMenui"
href="#"><span>Results</span></a><!--[if lte IE 6]><table><tr><td><![endif]-->
   <ul class="pureCssMenum">
     <li class="pureCssMenui"><a class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultssummary">Summary</a></li>
     <li class="pureCssMenui"><a class="pureCssMenui"
href="#"><span>Protein add-ons</span></a><!--[if lte IE 6]><table><tr><td><![endif]-->
       <ul class="pureCssMenum">
         <li class="pureCssMenui"><a
class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultssolublezfp">Soluble

ZFPs</a></li>

         <li class="pureCssMenui"><a
class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultstightbindingzfp">Tight

binding ZFPs</a></li>

         <li class="pureCssMenui"><a
class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultsfunctionalizedzfp">Functionalized

ZFPs</a></li>

         <li class="pureCssMenui"><a
class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/protdnahybrid">Protein-DNA

origami hybrid</a></li>

       </ul>

<!--[if lte IE 6]></td></tr></table></a><![endif]--></li>

     <li class="pureCssMenui"><a class="pureCssMenui"
href="#"><span>Vertical stacks</span></a><!--[if lte IE 6]><table><tr><td><![endif]-->
       <ul class="pureCssMenum">
         <li class="pureCssMenui"><a
class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultsdnatethers">DNA

tethers</a></li>

         <li class="pureCssMenui"><a
class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultsproteintethers">Protein

tethers</a></li>

       </ul>

<!--[if lte IE 6]></td></tr></table></a><![endif]--></li>

   </ul>

<!--[if lte IE 6]></td></tr></table></a><![endif]--></li>

 <li class="pureCssMenui"><a class="pureCssMenui"
href="#"><span>Discussion</span></a><!--[if lte IE 6]><table><tr><td><![endif]-->
   <ul class="pureCssMenum">
     <li class="pureCssMenui"><a class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/discussion">Discussion</a></li>
     <li class="pureCssMenui"><a class="pureCssMenui"
href="#"><span>Applications</span></a><!--[if lte IE 6]><table><tr><td><![endif]-->
       <ul class="pureCssMenum">
         <li class="pureCssMenui"><a
class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/applabonchip">Lab-on-a-nanochip</a></li>
         <li class="pureCssMenui"><a
class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/appbiosynthteticcompartments">Biosynthetic

compartments</a></li>

         <li class="pureCssMenui"><a
class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/appbiosensors">Biosensors</a></li>
         <li class="pureCssMenui"><a
class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/appnanoelectronics">Nanoelectronics</a></li>
       </ul>
     </li>
   </ul>

<!--[if lte IE 6]></td></tr></table></a><![endif]--></li>

 <li class="pureCssMenui"><a class="pureCssMenui"
href="#"><span>Methods</span></a><!--[if lte IE 6]><table><tr><td><![endif]-->
   <ul class="pureCssMenum">
     <li class="pureCssMenui"><a class="pureCssMenui"
href="#"><span>DNA origami design</span></a><!--[if lte IE 6]><table><tr><td><![endif]-->
       <ul class="pureCssMenum">
         <li class="pureCssMenui"><a
class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methgeneraldesignstrategy">General

design strategy</a></li>

         <li class="pureCssMenui"><a
class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methmodifications%20">Modifications</a></li>
       </ul>

<!--[if lte IE 6]></td></tr></table></a><![endif]--></li>

     <li class="pureCssMenui"><a class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methplasmidconst">Plasmid

construction</a></li>

     <li class="pureCssMenui"><a class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methprotprodandisol">Protein

production and isolation</a></li>

     <li class="pureCssMenui"><a class="pureCssMenui"
href="#"><span>Protein characterization</span></a><!--[if lte IE 6]><table><tr><td><![endif]-->
       <ul class="pureCssMenum">
         <li class="pureCssMenui"><a
class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methemsa">EMSA</a></li>
         <li class="pureCssMenui"><a
class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methalphascreen">AlphaScreen

assay</a></li>

         <li class="pureCssMenui"><a
class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methbret">BRET

assay</a></li>

       </ul>

<!--[if lte IE 6]></td></tr></table></a><![endif]--></li>

     <li class="pureCssMenui"><a class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methafm">AFM</a></li>
     <li class="pureCssMenui"><a class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methtimeline">Timeline</a></li>
   </ul>

<!--[if lte IE 6]></td></tr></table></a><![endif]--></li>

 <li class="pureCssMenui"><a class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/team">Team</a></li>
 <li class="pureCssMenui"><a class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/acknowledgments">Acknowledgments</a></li>

</ul> <!-- End PureCSSMenu.com MENU --> </div> </div> </div> <!-- end #header --> <div id="page" class="container"> <div id="content"> <div class="post"> <div class="entry"><big><big><big><big><span

style="color: black; font-weight: bold;">Protein tethers</span></big></big></big></big><br>

<br><br> <span style="font-family: Arial;"> In addition to vertical stacking of DNA origami layers using ssDNA we thought of proteins as a tool for achieving the same goal. In particular protein tethering could precisely define the distance between the functionalized DNA origami layers. Many different combinations of distinct twin ZFP chimeras can be rationally designed based on the library of characterized ZFPs (ZiFDB). It is also possible to engineer ZFPs from scratch, especially since the "context-dependent assembly" or CoDA was introduced in 2010 (Sander, 2011), increasing the efficiency of this approach. </span><br style="font-family: Arial;"> <br style="font-family: Arial;"> <span style="font-family: Arial;">As described in the Idea section twin ZFP chimeras could be used to tether DNA origamis into vertical stacks through interactions with modified binding staples extending perpendicularly from the DNA origami. We constructed twin ZFP chimeras in the form of ZFP1 - MBP - ZFP2, where two distinct ZFPs are attached to both termini of the solubility tag MBP. </span><br> <br style="font-family: Arial;"> <table

style="margin-top: 3px; margin-bottom: 20px; float: left; width: 100%; height: 50px;"
border="0" cellpadding="0" cellspacing="0">
 <tbody>
   <tr>
     <td style="text-align: center;"><img
style="font-family: Arial; width: 890px; height: 298px;" alt=""
src="http://openwetware.org/images/d/d8/ProteintetheringFINAL.png"></td>
   </tr>
   <tr style="font-family: Arial;">
     <td style="text-align: justify;"><span
style="font-weight: bold;">Figure 35. Protein-based tethering

of DNA origami planes with twin ZFP chimeras. </span>(a) Basic element for such protein mediated tethering comprises a solubility enhancing domain (MBP) flanked by two distinct ZFPs. (b) Numerous recombinantly produced twin ZFP chimeras stabilize DNA origami planes at defined distance locking down the stack.</td>

   </tr>
 </tbody>

</table> <br style="font-family: Arial;"> <span style="font-family: Arial;">We designed and successfully isolated two such twin-ZFP chimeras, namely 2C7-MBP-6F6 and AZPA4-MBP-6F6. Since MBP acted as a solubility tag we could thus observe high production of soluble protein at the expected size in the cell lysate. However insoluble fraction contained some of the protein, probably due to the presence of two ZFPs domains in the fusion protein. We purified both twin ZFP chimeras, which will be used to prepare protein-tethered DNA origami vertical stacks. The results of the proteins' production, confirmation of their identity by Western blot and purification are presented below.</span><br> <br style="font-family: Arial;"> <table

style="margin-top: 3px; margin-bottom: 20px; float: left; width: 100%; height: 50px;"
border="0" cellpadding="0" cellspacing="0">
 <tbody>
   <tr>
     <td style="text-align: center;"><img
title="http://openwetware.org/images/6/61/TwinZFPsProductionandisolation.png"
style="margin-bottom: 10px; font-family: Arial; width: 890px; height: 328px;" alt=""
src="http://openwetware.org/images/6/61/TwinZFPsProductionandisolation.png"></td>
   </tr>
   <tr style="font-family: Arial;">
     <td style="text-align: justify;"><span
style="font-weight: bold;">Figure 36: Production, WB

confirmation and isolation of two distinct twin ZFP chimeras. </span>(a) Coomassie Brilliant Blue stain of the soluble and insoluble fractions of the bacterial cell lysate after protein production. (b) Western blot of fractions in the panel (a) using anti-His antibodies. (c) Coomassie Brilliant Blue stain of the twin ZFP proteins purified using chelating chromatography. Grey arrow depicts the position of 2C7-MBP-6F6 (Mr of 86 kDa) and the black arrow the position of AZPA4-MBP-6F6 (Mr of 84 kDa). Proteins were produced under the same conditions as BRET triple fusions: 2x YT medium supplemented with 10 g/L glucose, 100 mg/L antibiotic kanamycin and 0.5 mM ZnCl<sub>2</sub> / 30 °C or 37 °C / 160rpm / ~5 or 7 h induction with 1 mM IPTG.</td>

   </tr>
 </tbody>

</table> <br style="font-family: Arial;"> <span style="font-family: Arial;">Previous demonstration of the specific binding of GST-AZPA4 protein chimera to a rectangular DNA origami (Tight binding ZFPs, AFM) strongly suggests the isolated twin ZFPs will be able to form hybrid 3D DNA-protein nanostructures.</span><br> <br style="font-family: Arial;"> <hr style="width: 100%; height: 2px;"> <small> <ul>

 <li>Sander JD, Dahlborg EJ, Goodwin MJ, Cade L, Zhang F,

Cifuentes D, Curtin SJ, Blackburn JS, Thibodeau-Beganny S, Qi Y, Pierick CJ, Hoffman E, Maeder ML, Khayter C, Reyon D, Dobbs D, Langenau DM, Stupar RM, Giraldez AJ, Voytas DF, Peterson RT, Yeh JR, Joung JK (2011) Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nature Methods 8: 67-69.</li> </ul> </small> </div> </div> </div> <!-- end #content --> <div style="clear: both;">&nbsp;</div> </div> <!-- end #page --></div> <div id="footer-content" class="container"> <div id="footer-bg"> <table

style="width: 100%; text-align: left; margin-left: auto; margin-right: auto;"
border="0" cellpadding="2" cellspacing="2">
 <tbody>
   <tr>
     <td style="text-align: right;"><a
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultsdnatethers"><img
alt="" style="border: 0px solid ; width: 132px; height: 39px;"
src="http://openwetware.org/images/d/d3/Previousblue.JPG"></a></td>
     <td style="text-align: left;"><a
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/discussion"><img
alt="" style="border: 0px solid ; width: 132px; height: 39px;"
src="http://openwetware.org/images/6/69/Nextblue.JPG"></a></td>
   </tr>
 </tbody>

</table> </div> </div> <div id="footerCSS"> <p>BioNanoWizards - BioMod 2011 team Slovenia. Design by <a href="http://www.freecsstemplates.org/">Free CSS Templates</a>.</p> </div> <!-- end #footer --> </body> </html>