Biomod/2012/UT/Nanowranglers/Results

From OpenWetWare
Revision as of 17:08, 27 October 2012 by Ben Braun (talk | contribs) (Future Work)
Jump to: navigation, search

<html> <style type="text/css"> //<!--

//Get the playbill font by Hank Gillette, 1992, public domain @font-face {

 font-family: Playbill2;  
 src: url( 'http://openwetware.org/images/e/e6/PLAYBILL.eot' ); /* IE */  
 src: local(Playbill), 
      url( 'http://openwetware.org/images/a/a1/PLAYBILL.TTF' ) format('truetype'),
      url( 'http://openwetware.org/images/b/ba/PLAYBILL.svg#font' ) format('svg'); /* non-IE */  

} /* Define the background of the whole page */ @media screen {

   body { background: #c5d0ea 0 0 no-repeat; }

} /* Define the div holding the page contents */

  1. globalWrapper{
 left: 50%;
 height: 100%;
 background-color: #FFFFFF;
 width: 900px;
 margin-left: -450px; /* Should be width / 2 */
 margin-top: 0px;
 margin-bottom: 0px;
 margin-right: 0px;
 padding: 0 0 0 0;   
 position: absolute;

} .toc{

 display:none;

} /* Hide the page heading at top */ .firstHeading {display:none;} /* Hide the bar at the left */

  1. column-one {display:none;}

/* Define the div holding the page contents (this div is inside globalWrapper. We override its default margins) */

  1. content{
 margin: 0 0 0 0;
 font: normal 100% sans-serif;
 color: #000033;
 padding: 12px 12px 12px 12px;
 background-image: url(http://openwetware.org/images/3/38/FortDavis-Pano-Cropped.jpg); background-repeat: no-repeat; 
 border: 0;

} /* Miscellaneous stuff. Feel free to add style definitions. */ h3 {

 /*
 color: #CC5500;
 font: normal 3em sans-serif;
 letter-spacing: 1px; 
 margin-bottom: 0; 
 */
 color: #000000;
 font-style: normal;
 font-size: 39px;
 font-family: 'Playbill2', 'Playbill', sans-serif;
 //font-stretch: ultra-expanded;
 letter-spacing: 1px; 
 margin-bottom: -10px;
 line-height: 25px;
 padding-bottom: 0px;

} h2 {

 color: #000000;
 font-style: normal;
 font-size: 39px;
 font-family: 'Playbill2', 'Playbill', sans-serif;
 //font-stretch: ultra-expanded;
 letter-spacing: 1px; 
 margin-bottom: -10px;
 line-height: 25px;
 padding-bottom: 0px;

} h1 {

 /*
 color: #CC5500;
 font: normal 3em sans-serif;
 letter-spacing: 1px; 
 margin-bottom: 0; 
 */
 color: #000000;
 font-style: normal;
 font-size: 39px;
 font-family: 'Playbill2', 'Playbill', sans-serif;
 //font-stretch: ultra-expanded;
 letter-spacing: 1px; 
 margin-bottom: -10px;
 line-height: 25px;
 padding-bottom: 0px;

}

.scriptFont {

  color: #0;
  padding-top: 5px;
  font: normal 1em Georgia;

}

/* Code taken from publicly available css tutorials at onderhond.com */ .linkconcept a {background:url("http://openwetware.org/images/0/03/Concept-bright.png") left top no-repeat;} .linkmembers a {background:url("http://openwetware.org/images/2/24/Members-bright.png") left top no-repeat;} .linkresults a {background:url("http://openwetware.org/images/a/ac/Results-bright.png") left top no-repeat;} .linkmethods a {background:url("http://openwetware.org/images/0/02/Methods-bright.png") left top no-repeat;} .linkrefs a {background:url("http://openwetware.org/images/2/28/References-bright.png") left top no-repeat;}

.linkgeneral {float:left; padding-right:3px; } .linkgeneral a {display:block; width:141px; height:52px;} .linkgeneral a:hover img {position:absolute; left:-999em; top:-999em;} .linkgeneral a:hover {zoom:1;} //--> </style> <div style="text-align:center" /> <img src="http://openwetware.org/images/5/59/NanoWranglers.png"/><br /> <div class="scriptFont"> Undergraduate DNA nanotechnology research group from the University of Texas at Austin </div> <img src="http://openwetware.org/images/e/e6/NanoWranglersDNAStripe.png"/><br /> <!-- links at top --> <div style="float:left; width:85px; height:52px; display:block" > </div> <div class="linkconcept linkgeneral">

 <a href="http://openwetware.org/wiki/Biomod/2012/UT/Nanowranglers"><img src="http://openwetware.org/images/c/ce/Concept-dull.png" alt="" /></a>

</div> <div class="linkmethods linkgeneral">

 <a href="http://openwetware.org/wiki/Biomod/2012/UT/Nanowranglers/Methods"><img src="http://openwetware.org/images/b/be/Methods-dull.png" alt="" /></a>

</div> <div class="linkresults linkgeneral">

 <a href="http://openwetware.org/wiki/Biomod/2012/UT/Nanowranglers/Results"><img src="http://openwetware.org/images/8/8f/Results-dull.png" alt="" /></a>

</div> <div class="linkmembers linkgeneral">

 <a href="http://openwetware.org/wiki/Biomod/2012/UT/Nanowranglers/Members"><img src="http://openwetware.org/images/4/47/Members-dull.png" alt="" /></a>

</div> <div class="linkrefs linkgeneral">

 <a href="http://openwetware.org/wiki/Biomod/2012/UT/Nanowranglers/References"><img src="http://openwetware.org/images/4/49/References-dull.png" alt="" /></a>

</div> <div style="clear: both;" ></div> <img src="http://openwetware.org/images/e/e6/NanoWranglersDNAStripe.png"/><br />

</div> </html>


Results

We used several methods to investigate the progress of our walker. We simulated the design using Kintek Explorer and this allowed us to determine the relative times for assembling and testing. Next we used electrophoresis gels to purify the strands and to analyze the design. What follows is a summary of those methods.

Kinetic simulation of catalyzed hairpin assembly

WalkerKineticsTable.jpg

The following is a screencapture of the Kintek output. Time in seconds is on the x-axis and concentration in μM is on the y-axis.

The initiator is shown in black, fuel 1 in red, fuel 2 in teal, and the duplexed fuel 1 and fuel 2 in yellow.

CHA kinetics graph.png

Electrophoresis Gels

To for the gel, it is cross-linked using 10% APS and TEMED between two glass plates, then clipped to an electrophoresis rig which uses voltage to drive the negatively charged DNA down the cross-linked polyacrylamide gel. As the strands of DNA are moving through the gel, the shorter ones will pass faster than longer ones. This means there is a separation of the DNA based entirely on molecular weight. If the DNA is pure enough, bands will be present in the fluorescent image of the gel. These bands can be correlated to a known standard, a DNA “ladder” that contains specific sequence lengths, that is run alongside the samples. Using the number and location of the bands allows us to reach conclusions about how our design is working. We chose to test two buffer conditions for our system that are specifically designed to help large DNA structures form and maintain stability.

Walker System Test, 10-27-2012 - Buffer: TMgK


File:Walker system 10-27-2012.tif

Walker system 10-27-2012 table.png


Fuel Test 1, 8-13-2012 - Buffer: TMgK


File:8-13-2012TMgKgel.jpeg8-13-2012TMgKtable.jpeg


Conclusion: There is significant hairpin leakage between FA,h and FB,h, and also between FA,h and FD,h, but this decreases noticeably in the absence of the initiator. From the presence of excess lines in both lanes that contain FD,h, it is clear that there are impurities in FD,h, which will require the test to be run again after the sample is purified.


Fuel Test 2, 8-14-2012 - Buffer: TMgK


8-14-2012TMgKgel.jpeg8-14-2012TMgKtable.jpeg


Conclusion: There is significant hairpin leakage between FA,h and FB,h, and also between FA,h and FD,h, regardless of whether the initiator is present. While this trend does decrease slightly when temperature of incubation is decreased and concentration of FA,h is increased, the change is not dramatic enough to make these hairpins viable as fuels in this buffer.


Fuel Test 2, 8-14-2012 - Buffer: TNaK


8-14-2012TNaKgel.jpeg8-14-2012TNaKtable.jpeg


Conclusion: There is significant hairpin leakage between FA,h and FB,h, and also between FA,h and FD,h, regardless of whether the initiator is present. There is no noticeable change in this trend when temperature of incubation is decreased or concentration of FA,h is increased. Therefore, these hairpins are not viable as fuels in this buffer.

Future Work

We have shown:

  • The assembly of the walker system using acrylamide gel
  • The functionality of the CHA fuel system using kinetic simulation and fluorometric measurements

We will continue working to build this walker system. To do this we need to verify walker functionality using fluorometric measurements, calibrate the results for leakage, and determine optimum buffer conditions. The nicking enzyme will be tested using an acrylamide gel to determine validity. We also will work to redesign the fuel strands so the walker is capable to reverse its direction.