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<div><img src="http://openwetware.org/images/5/5d/Barrel_Saxs_Models.png" /></div>
<div><img src="http://openwetware.org/images/5/5d/Barrel_Saxs_Models.png" /></div>
Fig 4. Extremes of 3D model scan for the barrel.
Fig 4. Extremes of 3D model scan for the barrel.
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<div><img src="http://openwetware.org/images/e/ec/Coop_Saxs_Models.png" /></div>
Fig 5. 3D model of expected cooperative switch structure.
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<div id="LAB-BOOK-TOP"> <div id="LAB-BOOK-TITLE" style="padding-left:60px; text-align: justify;">Experiment 4 - Characterisation of Cooperative Biosensor</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> <div id="LAB-BOOK-TEXT">

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<h4> Synthesis of Core Barrel and Cooperative biosensor</h4> <h2>Aim</h2> To see whether can build the Core barrel of our cooperative biosensor, and the whole biosensor as designed.

<h2>Background</h2> Having designed and generated the sequences required for our<a href="http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Coop"> core barrel and cooperatively linked biosensor </a> we needed to build it. 

				<h2>Methods and Materials</h2>

After generating sufficiently high-yields of our <a href="http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Exp3">custom DNA scaffold </a>, we confirmed the purity on an agarose gel and estimated the concentration by Nanodrop. <br> <br> At the same time, we combined all our synthesised oligos into a common mix wherever possible. This meant we used: <br><br> <ul> <li>a staple mix (12 oligos) to assemble the barrel at the core of our biosensor, </li> <li>a sensor mix (36 oligos) to assemble the switches on the outside of our barrel, </li> <li>three band mixes (24 oligos each) to assemble the different cooperative biosensors with ostensibly different functionality</li> </ul> <br> These mixes were created by adding 2ul of each oligo at 500µM into their common mix. This was mixed thoroughly by pipetting and centrifuged at ~20,000g for 30 minutes to pellet any aggregate. The supernatant was removed and the concentration estimated by Nanodrop. <br><br> We then calculated the volumes of the scaffold and various staple mixes required to synthesise the biosensor at a particular concentration depending on the experiment required. For example Small Angle X-ray Scattering requires a high concentration of about 500ng/uL of sensor. We combined the scaffold and staples in a single PCR tube and synthesised them on a thermocycler by heating to 90˚C and cooling back to room temperatature by 0.5˚C/min.<br><br> We initially assembled the Core Barrel structure. To get some indication that it had indeed circularised to form a barrel, we included a synthesis of a 'broken' barrel that was missing a set of staples joined two ends of the barrel.

<h2>Results</h2> We demonstrated that our core barrel could be assembled (see Fig. 3.) using our custom scaffold and that an annealing step was necessary to achieve self-assembly of our structure in a reasonable time frame. Additionally, the full and partial synthesis constructs ran differently through an agarose gel, indicating that they had different structures. <br><br> <div class="image-center"> <div><img src="http://openwetware.org/images/4/49/2014-EchiDNA-LAB-BOOK-EXPERIMENT_4-shit_Fig1.png" /></div> Fig 3. The final two wells of this gel show the assembled barrel at the core of of cooperative biosensor. 'Neat' indicates what happens when the staples and added to the scaffold and left at room temperature, whereas 'anneal' shows what happens after heating and cooling the mixture. Note that the two types of barrel run to a slightly different length, which indicates what happens when we change a 'barrel' into a 'tile' by removing those staples that connect the opposite ends of our scaffold strand. </div> <br><br> <h4>Purification of Cooperative biosensor</h4> <h2>Aim</h2> To purify the assmegled biosensor away from staples

<h2>Background</h2> Yields of DNA origami structures are maximised when a large, single scaffold strand is the limiting part, and an excess of staple strands are used to fold it. The use of a single limiting strand that traverses the entirety of the desired structure and a vast excess of smaller strands reduces the possibility of partially-assembled structures using up all the material. This synthesis strategy results in fully assembled structures an a large amount of waste material. For further studies, the excess staples must be removed as they would add extra noise in structural analyses and the presence excess ’sensor strands' would confound our functional analyses of the cooperative biosensor. <br> 

				<h2>Methods and Materials</h2>

We first tried purifying our constructs using a <a href="http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_17395/17059901">sephacryl S-300 High Resolution gel matrix</a>. <br><br> We loaded the S300 bead slurry in to 3x empty micro-centrifuge spin columns. The beads were equilibrated with 5 column volumes of synthesis buffer (1000g 1 min) and spun dry 1000g 4 min). <br><br> 50µL of un-purified assembly was loaded onto the dry columns and spun through (1000g 1 min). This flow through loaded onto another column and repeated 5 times. <br><br> 

				<h2>Results</h2>
Although, this purification protocol has previously been successful for other DNA nano-structures with 32nt staples , since the size exclusion limit of this gel was ~118bp while the largest of our staples was 86nt and in excess, we were not able to separate these long staples. :(

<br><br> <div class="image-center"> <div><img src="http://openwetware.org/images/a/a5/2014-EchiDNA-LAB-BOOK-EXPERIMENT_4-shit_Fig4.png" /></div> Fig 4. We showed that despite the size exclusion limit of S-300 beads being twice the mass of our largest staples, we still couldn't completely purify our staples using sephracryl S-300. </div> <br><br>


<h4>Small Angle X-ray Scattering</h4>

<h2>Aims</h2> To use Small Angle X-ray Scattering to experimentally acquire basic structural information about the Cooperative sensor <h2>Background</h2> We were very fortunate to receive sponsorship from the <a hrehttp://www.synchrotron.org.au/">Australian Synchrotron </a> who provided us with the ability to carry out.Small-Angle X-ray Scattering (SAXS) experiments for the structural characterisation of our Cooperative Biosensor.

Small angle x-ray scattering is a technique which provides utilises the small wavelength of x-rays to provide information on the nanometer scale about the structure of materials. When x-rays interact with electrons in the material of the sample, their path is bent or "scattered" and from the resulting pattern. By looking at this diffraction at small angles (of less than 10 degrees) it is possible to calculate the relative distribution of the atoms in the material. SAXS has a key advantages over other structural techniques such as x-ray crystallography as it does not require the material being investigated to be crystalline, and AFM and EM as the structure is in solution rather than fixed on a surface. <br><br>

<h2>Methods and Materials</h2> In order to prepare a sample for SAXS, a concentration of ideally 500 ng/uL is required with higher concentrations yielding stronger, more easily analysed signals. We synthesised and purified barrel and cooperative sensor samples as described above.

To analyse the scatter pattern we produced a 3D model defining locations of hydrogen-bonded base-pair in each helix as we expected them scanned a range of spacings between helices.

<br><br> <div class="image-center"> <div><img src="http://openwetware.org/images/5/5d/Barrel_Saxs_Models.png" /></div> Fig 4. Extremes of 3D model scan for the barrel. </div>

<div class="image-center"> <div><img src="http://openwetware.org/images/e/ec/Coop_Saxs_Models.png" /></div> Fig 5. 3D model of expected cooperative switch structure. </div>

<h2>Results</h2>

<h2>Conclusion</h2>

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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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