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Fig 1. Overview of reaction with the first design of the single beacon.
Fig 1. Overview of reaction with the first design of the single beacon.
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We actually iterated through this set of experiments twice, covering two evolving versions of our re-designed molecular beacon.  
We actually iterated through this set of experiments twice, with two evolving versions of our molecular beacon.
 
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<div><img src="http://openwetware.org/images/a/a5/2014-EchiDNA-EXP2-DESIGN-2.png"  /></div>
<div><img src="http://openwetware.org/images/a/a5/2014-EchiDNA-EXP2-DESIGN-2.png"  /></div>
Fig 2. Overview of reaction with the second design of the single beacon.
Fig 2. Overview of reaction with the second design of the single beacon.
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Through these experiments we found that our first design had <a href="
http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Single" target="_blank">two major problems</a>, which we managed to overcome in the second design.
 
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Revision as of 18:08, 25 October 2014

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<div id="LAB-BOOK-TOP"> <div id="LAB-BOOK-TITLE" style="padding-left:60px; text-align: justify;">Experiment 2 - Characterisation of Molecular Beacon</div> </div> <div id="LAB-BOOK-REPEAT"> <img src="http://openwetware.org/images/8/81/2014-EchiDNA-LAB-BOOK-EXPERIMENT-CLEAN-BOOK.png" /> <a href="http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook" id="LAB-BOOK-CLEAN-BOOK"></a> <a href="http://biomodaustralia2014.postach.io/" id="LAB-BOOK-DIRTY-BOOK" target="_blank"></a>

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<h2>Aim</h2> To build and characterise a single non-cooperative switch, and specifically to: <ul><li> optimise purity and yield of switch construction,</li> <li> and investigate the sensitivity of molecular beacons by systematically varying clip strength and signal concentration.</li></ul>

<h2>Background</h2>

We <a href="http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Single" target="_blank">re-designed</a> conventional molecular beacons to suit them to a new application in our cooperative molecular biosensor. We needed to determine whether our stripped-down, modular version of molecular beacons really worked! <br><br> There were three key measures of whether our re-designed molecular beacons were a success: <ul> <li>Firstly, we had to see whether or not our de-constructed version of the single-stranded molecular beacon would still hybridise into a complex of four separate oligos with predictable efficiency. </li> <li> Secondly, we had to determine whether the re-designed molecular beacon still functioned and identify the limits of the switch in terms of signal-to-noise and the dynamic range of biosensing activity. </li> <li> Finally, we wanted to exploit the modularity of our design to fully explore and characterise the potential of the switch as a biosensing technology. </li> </ul> In the first case one of the simplest and most direct ways to observe assembly is simply to create samples where all the components of the molecular biosensor are isolated, and others where they are together. By applying an electric field over a non-denaturing poly-acrylamide gel (PAGE) containing the DNA samples, it was possible to directly observe the change in mass after mixing complementary oligos. It was also possible to observe the oligo-bound fluorophore changing in mass upon hybridisation. In the second and third cases, we moved directly towards fluorescence as a direct means of testing whether the redesigned molecular beacon worked as anticipated. <br> <br> <div class="image-right"> <div><img src="http://openwetware.org/images/1/1d/2014-EchiDNA-EXP2-DESIGN-1.png" /></div> Fig 1. Overview of reaction with the first design of the single beacon. </div> We actually iterated through this set of experiments twice, with two evolving versions of our molecular beacon. <div class="image-right"> <div><img src="http://openwetware.org/images/a/a5/2014-EchiDNA-EXP2-DESIGN-2.png" /></div> Fig 2. Overview of reaction with the second design of the single beacon. </div> Through these experiments we found that our first design had <a href=" http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Single" target="_blank">two major problems</a>, which we managed to overcome in the second design.

<br>

<div class="image-center"> <div><img src="http://openwetware.org/images/d/d6/2014-EchiDNA-EXP2-CLIP-STRENGTH.png" /></div> Fig 3. Explanation of different clip strengths. </div><br>

<h2>Methods and Materials</h2> <orange> Confirmation of assembly via spontaneous hybridisation and annealing </orange><br> The completed assemblies were examined on gels and the fluorescent plate reader.<br><br>

<orange> Assembling the non-cooperative switch (excess vs molar ratios) </orange> <br>

<div class="image-right"> <div><img src="http://openwetware.org/images/9/91/PossibleSingleCombinations.png" /></div> Fig 4. An initial table of the 32 possible combinations of the individual 5 components. Note the assumption the flurophore will behave consistently except in the presence of a proximate quencher, which was later determined to be false <a href="http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Exp1"> (see experiment 1) </a> </div>


While molecular beacons can be as simple as a single strand of DNA, in order to rapidly investigate multiple tuneable systems on our non-cooperative cooperative switch, it was desirable to have a modular switch. However, the most immediate cost of having a system with independent oligos for each subcomponent of the switch is the large number of incomplete assemblies that are possible. <br>


The main cases we investigated were equivalent mole ratios of each component, slight excess of components relative to the longest strand (referred to as the backbone or scaffold) and large excess of components relative to the longest strand. <br><br>


<orange> Titration of switch against signal - "Grid experiments" </orange> <br> In characterising the behaviour of the single switch, we wanted to observe the resulting fluorescence from varying the signal concentration, clip strength, and exposure time. The result was a experiment where grid of fixed amount of switches of each <br><br>

<div class="image-center"> <div><img src="http://openwetware.org/images/thumb/0/0f/GridMethod1.jpg/336px-GridMethod1.jpg" /></div> Fig 5. Tables for construction of various switches and testing against varying signal concentration </div><br>

<h2>Results</h2> <orange> Construction Results </orange> <br> <br>

From the polyacrylamide gel, stained with SYBR Gold, we are clearly able to identify the distinct bands of each individual components of the switch.

<div class="image-center"> <div><img src="http://openwetware.org/images/1/1a/20140929wgold.png" /></div> Fig 6. Polyacrylamide gel demonstrating successful assembly of switch with high efficiency. Key: </div><br>


<orange> Grid Results </orange> <br>

<div class="image-center"> <div><img src="http://openwetware.org/images/b/b6/SingleSwitchGrid1.png" /></div> Fig 9. Tables for construction of various switches and testing against varying signal concentration </div><br>

<div class="image-center"> <div><img src="http://openwetware.org/images/3/36/SingleSwitchGrid2.png" /></div> Fig 10. Tables for construction of various switches and testing against varying signal concentration </div><br>


<div class="image-center"> <div><img src="http://openwetware.org/images/2/2a/Grid_over_time2.gif" /></div> Fig 11. Tables for construction of various switches and testing against varying signal concentration </div><br>


<h2>Conclusion</h2> We concluded that we were successfully able to construct a single modular non-cooperative DNA biosensor based on the principles of fluorescence. We observed from our gels a room temperature hybridisation that provided high yields and optimised our molar ratios for switch construction. We were able to observe the expected sigmoidal behaviour of our switch in response to varying concentrations of


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Contact: <a href="mailto:BIOMODaustralia@gmail.com">BIOMODaustralia@gmail.com</a> | ©2014 Team EchiDNA. All rights reserved. This team is supported by the <a id="vccri" href="http://www.victorchang.edu.au">Victor Chang Cardiac Research Institute</a>. | <a id="edit-link" href="">Edit</a>

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