Biomod/2011/Slovenia/BioNanoWizards/resultsfunctionalizedzfp: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
No edit summary
Line 658: Line 658:
     <tr>
     <tr>
       <td style="text-align: left;"><img
       <td style="text-align: left;"><img
  style="margin-right: 20px; width: 175px; height: 268px;" alt=""
  style="margin-right: 20px; width: 200px; height: 306px;" alt=""
  src="http://openwetware.org/images/6/63/BRETfusionsisolationCBB.png"></td>
  src="http://openwetware.org/images/6/63/BRETfusionsisolationCBB.png"></td>
       <td style="text-align: justify; vertical-align: top;"><span
       <td style="text-align: justify; vertical-align: top;"><span

Revision as of 20:15, 1 November 2011

<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;">Functionalized ZFPs</span></big></big></big></big><br>

<br> <span style="font-family: Arial;"> Protein domains exhibit a wide variety of physicochemical properties and represent an important pool for delivering the specific function to a defined position on DNA origami. In order to demonstrate the proof of the concept for spatial addressing of protein functions on a DNA origami, we considered using restriction enzymes, proteases, biosynthetic enzymes, proteins with optical properties, motor proteins etc.<br> &nbsp;</span><br style="font-family: Arial;"> <table 

style="margin: 3px 0px 20px 20px; width: 444px; height: 50px; float: right;"
border="0" cellpadding="0" cellspacing="0">
 <tbody>
   <tr>
     <td style="text-align: center;"><img
style="padding: 0pt 0pt 5px; width: 460px; height: 345px;"
alt="This is picture of origami rose" title="Origami rose"
src="http://upload.wikimedia.org/wikipedia/commons/thumb/6/61/Rosa_de_papel.jpg/1024px-Rosa_de_papel.jpg"></td>
   </tr>
   <tr style="font-family: Arial; font-weight: bold;">
     <td style="text-align: justify;">Figure.<span
style="font-weight: normal;"> Slika Modela BRET fuzij ali

shema delovanja BRETa (ni v sekciji metod)</span></td> 

   </tr>
 </tbody>

</table> <span style="font-family: Arial;">Finally we decided to explore the performance of BRET - bioluminescence energy transfer on the DNA origami. BRET is a biophysical phenomenon in which energy is non-radiatively transferred from the donor (Renilla luciferase in our case) to the acceptor (mCitrine variant of the yellow fluorescent protein) when both molecules are bound in close proximity (within 10 - 100Å range). Renilla luciferase (RLuc) catalyzes the reaction of oxidative decarboxylation converting its substrate coelenterazine to coelenteramide which emits light with a maximum emission peak at around 475 nm. The energy of photons is high enough to excite yellow fluorescent protein (in case it is bound within the required distance away from Renilla luciferase) leading to fluorescence with an emission maximum of 529 nm. Upon successful implementation of a BRET pair binding to DNA origami, the system could be expanded to four partners by splitting Renilla luciferase and mCitrine in 2 segments, where BRET would depend on the proximity of four sites (two pairs). Because of the distance-dependent signal efficiency, BRET could be employed as a nanomolecular ruler or as a logical operator for <a 

href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/appbiosensors">biosensors</a>

as outlined in the Applications section. </span><br style="font-family: Arial;"> <br style="font-family: Arial;"> <span style="font-family: Arial;">We first prepared constructs for BRET protein chimeras in the form of RLuc-2C7 and YFP-AZPA4. Unfortunately, it turned out the two proteins were expressed exclusively in the insoluble fraction of the cell lysate, meaning they were aggregated and would need refolding for binding to DNA origami. Several isolation and refolding attempts were tried with little success. </span><br style="font-family: Arial;"> <br style="font-family: Arial;"> <span style="font-family: Arial;">We tried to optimize the production conditions in order to purify the produced protein fusions in the native form. A well-known strategy for enhancing solubility and proper folding of larger protein fusions during their production is to lower the temperature as well as the concentration of the inducer IPTG giving the protein time to fold correctly and decrease the formation of protein aggregates in vivo. We achieved partial expression of soluble YFP-AZPA4 (Figure 2), with no improvement on solubilization of RLUC-2C7 fusion.</span><br style="font-family: Arial;"> <br> <table style="text-align: left; width: 100%;" border="0" 

cellpadding="8">
 <tbody>
   <tr>
     <td style="text-align: center;"><img
style="width: 276px; height: 374px;" alt=""
src="http://openwetware.org/images/1/1f/InsolubleBRETfusionsWBproduction.png"></td>
     <td style="text-align: center;"><img alt=""
src="http://openwetware.org/images/6/66/MBPYFPAZPA4alteredcond.png"></td>
   </tr>
   <tr>
     <td style="text-align: justify; vertical-align: top;"><span
style="font-weight: bold;">Figure

1. ZFP-luciferase and -YFP fusions are produced in bacteria in an insoluble form.</span> Figure shows Western blot after production of fusion proteins intended for the BRET assay. Arrows to the left indicate the position of bands representing BRET fusions without MBP solubility tag present in an insoluble form (IBs - inclusion bodies). Grey arrow depicts the position of YFP-AZPA4 (Mr of 48.4 kDa) and black arrow the position of RLuc-2C7 (Mr of 59.5 kDa). Production conditions: 2x YT medium supplemented with 0.1 mM ZnCl2 and 100 mg/L antibiotic kanamycin / 37 °C / 180 rpm / ~7 h induction with 2 mM IPTG. </td> 

     <td style="text-align: justify; vertical-align: top;"><span
style="font-weight: bold;">Figure

2. Production of soluble YFP-AZPA4 fusion by modification of production conditions.</span> Western blot after production of YFP-AZPA4 with altered fermentation conditions (LB medium supplemented with 0.5 mM ZnCl2 and 100 mg/L antibiotic kanamycin / 24 °C / 160 rpm / ~24 h induction with 0.5 mM IPTG). This made YFP-AZPA4 protein (Mr of 48.3 kDa) observable in the soluble form (depicted by the arrow to the right), but not to sufficient extent to isolate it. </td> 

   </tr>
 </tbody>

</table> <br style="font-family: Arial;"> <span style="font-family: Arial;">Since we have already observed a beneficial effect of enhanced solubility by the attachment of MBP and/or GST domains to the N-terminal end of zinc finger proteins (section <a 

href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultssolublezfp">Soluble

ZFPs</a>), MBP domain was added to the N-termini of both BRET fusion proteins, creating triple fusion proteins in the following form: MBP-RLuc-2C7 and MBP-YFP-AZPA4 with an aim to increase their solubility and foster the isolation of functional proteins under native conditions.</span><br style="font-family: Arial;"> <br> <span style="font-family: Arial;">We anticipated that lowering the temperature during the protein production would lead to further increase of the final protein titers but ultimately we observed the solubility of both triple chimeras was comparable at 30 °C and 37 °C as can be inferred from the results below. We used the following conditions for the production: 2x YT medium supplemented with 10 g/L glucose, 100 mg/L antibiotic kanamycin and 0.5 mM ZnCl2 / 30 °C or 37 °C / 160rpm / ~5 or 7 h induction with 1 mM IPTG.</span><br><br> <span style="font-family: Arial;"></span><br> <table style="text-align: left; width: 100%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
   <tr>
     <td style="text-align: left;"><img
style="margin-bottom: 10px; width: 890px; height: 430px;" alt=""
src="http://openwetware.org/images/0/01/BRETfusionswithsolubilityTagproductionCBB.png"></td>
   </tr>
   <tr>
     <td style="text-align: justify;" valign="undefined"><span
style="font-weight: bold;">Figure 3. ZFP-BRET-MBP fusions

are produced in soluble form.</span> Coomassie Brilliant Blue stain of bacterial lysate fractions after production of BRET fusions with MBP domain. Grey arrow to the right indicates the approximate position of MBP-YFP-AZPA4 (Mr of 90.4 kDa) and black arrow the position of MBP-RLuc-2C7 (Mr of 101.5 kDa). As anticipated, the addition of MBP solubility tag promoted the presence of both protein chimeras in supernatant (SN). Both proteins were partially present in inclusion bodies (IBs) which was the case with all other ZFP chimeras as well.</td> 

   </tr>
 </tbody>

</table> <br> <br> <table style="text-align: left; width: 100%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
   <tr>
     <td style="text-align: left;"><img
style="margin-bottom: 10px; width: 882px; height: 499px;" alt=""
src="http://openwetware.org/images/0/0e/BRETfusionswithsolubilitytagproductionWB.png"></td>
   </tr>
   <tr>
     <td style="text-align: justify;" valign="undefined"><span
style="font-weight: bold;">Figure 4. ZFP-BRET-MBP fusions

are produced in soluble form. </span>Western Blot after production of BRET fusions with MBP tag. Grey arrow to the right indicates the expected position of MBP-YFP-AZPA4 (Mr of 90.4 kDa) and black arrow the position of MBP-RLuc-2C7 (Mr of 101.5 kDa).<br><br></td> 

   </tr>
 </tbody>

</table> <span style="font-family: Arial;"><br> </span> <table style="text-align: left; width: 75%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
   <tr>
     <td style="text-align: left;"><img
style="margin-right: 20px; width: 200px; height: 306px;" alt=""
src="http://openwetware.org/images/6/63/BRETfusionsisolationCBB.png"></td>
     <td style="text-align: justify; vertical-align: top;"><span
style="font-weight: bold;">Figure 5. Isolation of BRET

fusions with MBP tag using chelating chromatography.</span> Coomassie Brilliant Blue (CBB) stain of isolated fractions. Isolation of both BRET triple fusion proteins (MBP-YFP-AZPA4, Mr of 90.4 kDa and MBP-RLuc-2C7, Mr of 101.5 kDa) resulted in a single protein band observed after staining the SDS-PAGE gel with CBB. However, the isolation efficiency of MBP-YFP-AZPA4 was approximately 10-times higher compared to MBP-RLuc-2C7 as observed when determining proteins' concentrations after isolation. </td> 

   </tr>
 </tbody>

</table> <span style="font-family: Arial;"><br> After protein isolation various functional assays were performed to analyse the effect of added protein domains at both N- and C- terminus of yellow fluorescent protein (mCitrine) and Renilla luciferase (RLuc) We determined the fluorescence of YFP fusions and decarboxylation enzymatic activity on the luciferase's substrate coelenterazine leading to light emission in case of luciferase.</span><br> <br> <br> <big><big><span style="color: black; font-weight: bold;">Characterization of MBP-YFP-AZPA4</span></big></big><br 

style="font-family: Arial;">

<br> <table style="text-align: left; width: 100%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
   <tr>
     <td style="text-align: left;"><img
style="margin-right: 20px; width: 450px; height: 223px;" alt=""
src="http://openwetware.org/images/7/72/MBPYFPAZPA4fluorescentspectrum.png"></td>
     <td style="text-align: justify; vertical-align: top;"><span
style="font-weight: bold;">Figure 6. Fluorescence emission

spectrum of MBP-YFP-AZPA4 fusion. </span> Using PerkinElmer LS55 Luminescence Spectrometer the fluorescence emission spectrum of MBP-YFP-AZPA4 chimera was acquired. 130 μl of the supernatant was transferred to Hellma QS 3 mm quartz cuvette and excited at 485 nm. Emission spectrum was obtained within the 500 to 600 nm emission window with a scanning speed of 100 nm/min. Excitation and emission slits were adjusted to 5 nm. Red line represents blank control (lysis buffer), green line is a negative control (2C7-MBP-6F6 supernatant) and blue line shows the fluorescence emission spectrum of MBP-YFP-AZPA4 chimera.</td> 

   </tr>
 </tbody>

</table> <br> <br> <table style="text-align: left; width: 80%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
   <tr align="center">
     <td><img
style="margin-bottom: 10px; width: 750px; height: 254px;" alt=""
src="http://openwetware.org/images/2/23/MBPYFPAZPA4visual.png"></td>
   </tr>
   <tr align="justify">
     <td valign="undefined"><span
style="font-weight: bold;">Figure 7. Visual characterization

of the isolated MBP-YFP-AZPA4 chimera under UV light. </span> Left: centrifuge tubes with isolated MBP-YFPA-AZPA4 (50, 100 and 250 mM imidazole elution fractions) were illuminated with UV light. Negative control (isolated AZPA4-MBP-6F6) was added to the left of the three fluorescent samples to signify the difference in proteins optical properties. Right: 200 μl of 100mM imidazole elution fraction of MBP-YFP-AZPA4 was pippeted into Corning Costar 96-well white microtiter plate with transparent bottom to yield a pattern of "YFP". The plate was put into DNA Bio-Imaging Systems box and illuminated with the UV light.</td> 

   </tr>
 </tbody>

</table> <br> <br> <big><big><span style="color: black; font-weight: bold;">Characterization of MBP-RLuc-2C7<br> </span></big></big><span 

style="font-family: Arial;"><br>

<table style="text-align: left; width: 100%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
   <tr>
     <td style="text-align: left;"><img
style="margin-right: 20px; width: 400px; height: 307px;" alt=""
src="http://openwetware.org/images/2/21/MBPRLuc2C7biolum.png"></td>
     <td style="text-align: justify; vertical-align: top;"><span
style="font-weight: bold;">Figure 8. Bioluminescence

measurement for MBP-RLuc-2C7 chimera. </span> 40 μl of MBP-RLuc-2C7 supernatant (non-diluted, diluted 2 or 5-fold) was transferred to Corning Costar 96-well white microtiter plate. The bioluminescence assay was performed as follows using Berthold's ORION II Microplate Luminometer. After putting the microplate into luminometer 100 μl of 8 μM substrate coelenterazine h (Synchem) was injected into each well followed by photon emission as a result of luminescence, which was collected by the instrument for 1 sec and converted to RLU (relative luminescence units).</td> 

   </tr>
 </tbody>

</table> <br> <table style="text-align: left; width: 80%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
   <tr align="center">
     <td><span style="font-family: Arial;"><img
style="margin-right: 20px; width: 780px; height: 255px;" alt=""
src="http://openwetware.org/images/8/83/MBPRLuc2C7visual.png"></span></td>
   </tr>
   <tr align="justify">
     <td valign="undefined"><span
style="font-weight: bold;"></span><span
style="font-family: Arial;"><span
style="font-weight: bold;">Figure 9. Visual characterization

of the activity of MBP-RLuc-2C7 chimera. </span> Left: 100 μl of MBP-RLuc-2C7 supernatant was transferred to Perkin Elmer OptiPlateTM 96-well white plate in a pattern giving rise to the word "Luc". Right: After adding 100 μl of the coelenterazine h substrate solution prepared in the same way as for the bioluminescence assay with ORION II, the luminescence was collected in Syngene G:Box for 5 sec. Since Renilla luciferase exhibits fast reaction kinetics under these buffer conditions, some spots are brighter than others which is due to the fact that the substrate was not added into all microtiter wells simultaneously.<br><br></span></td> 

   </tr>
 </tbody>

</table> <br> <br> </span><big><big><span 

style="color: black; font-weight: bold;">BRET on DNA target</span></big></big><span
style="font-family: Arial;"><br>

<br> <table style="text-align: left; width: 80%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
   <tr align="center">
     <td><span style="font-family: Arial;"><img
style="margin-right: 20px; width: 780px; height: 133px;" alt=""
src="http://openwetware.org/images/3/32/BRETDNAtarget.png"></span></td>
   </tr>
   <tr align="justify">
     <td valign="undefined"><span
style="font-weight: bold;"></span><span
style="font-family: Arial;"><span
style="font-weight: bold;">Figure 10: Sequence of the BRET

DNA target. </span> 43 bp DNA target used to measure the BRET effect was designed as shown above. Blue colored is the 2C7 binding sequence and depicted in green is AZPA4 binding site. 2 bp long spacer was selected to separate the binding sites and flank of the same length at both 5' and 3' ends.<br><br></span></td> 

   </tr>
 </tbody>

</table> <br> <table style="text-align: left; width: 100%;" border="0" 

cellpadding="0" cellspacing="0">
 <tbody>
   <tr>
     <td style="text-align: left;"><img
style="margin-right: 20px; width: 400px; height: 260px;" alt=""
src="http://openwetware.org/images/b/b8/BRETgraphDNA.png"></td>
     <td style="text-align: justify; vertical-align: top;"><span
style="font-weight: bold;">Figure 11: BRET assay of the

mixture of chimeric ZFP fusions. </span> mCitrine emission was normalized to the 535 nm emission of the MBP-RLuc-2C7 without other protein or DNA components. Since the emission spectra of RLuc and mCitrine overlap, 535 nm emission peak is observed for 100nM MBP-RLuc-2C7 alone in the sample as well. When both BRET partners in 100 nM concentration are present and no DNA 535 nm emission peak increases possibly due to the random collisions between the molecules in the system. However, the addition of 50 nM DNA target furtherly increased mCitrine emission indicating the proximal binding of both fusions to the DNA target.</td> 

   </tr>
 </tbody>

</table> <br> We demonstrated the design, production, purification and characterization of the two protein elements required for the reconstitution of the BRET sensor on a DNA origami. Initial experiments support the functional BRET, however the overlapping Renilla luciferase and mCitrine emission spectra suggest that selection of other BRET partners might improve the signal to background ratio, e.g. RLuc8-GFP2 BRET pair as a reasonable choice (De, 2007). Sensitivity of the BRET effect on DNA origami could be improved by mounting several neighboring BRET pairs on a single DNA origami rectangle.<br> <br> </span> <hr style="width: 100%; height: 2px;"> <ul> 

 <li><small>De A, Loening AM,

Gambhir SS. (2007) An Improved Bioluminescence Resonance Energy Transfer Strategy for Imaging Intracellular Events in Single Cells and Living Subjects. <span style="font-style: italic;">Cancer Research</span>, 67: 7175-7183.</small><!-- TU SE KONEA GLAVNO BESEDILO NA STRANI --></li> </ul> </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/resultstightbindingzfp"><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/protdnahybrid"><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>

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Biomod/2011/Slovenia/BioNanoWizards/resultsfunctionalizedzfp&oldid=559980"