Biomod/2014/ASU/Results: Difference between revisions

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<p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">Testing Tile</span></p>

<p style = "color: #000000; font-size: 16px;">As a form of positive control, we created tiles of our basic design that could incorporate four random aptamers to specifically incorporate two aptamers known to bind alpha thrombin, called “aptamer A” and “aptamer B”, in specific configurations (shown below). The tiles were created using the tile assembly protocol under the Protocols tab of the Experiment section.</p><br>

<img src = "http://openwetware.org/images/8/8c/Screen_Shot_2014-10-23_at_4.05.45_AM.png">

<img src = "http://openwetware.org/images/c/c3/Screen_Shot_2014-10-23_at_4.06.24_AM.png">

<p style = "color: #000000; font-size: 16px;">After the above tiles were created, they were incubated at room temperature with alpha thrombin protein at a ratio of 20nM tile: 1uM protein for one hour with gentle rotation. The samples were then run on an 8% Native PAGE gel with 10x bromophenol blue at 200V for 8 hours at 20 degrees Celcius with stirring. The gel was stained with Sybr Green and imaged by a Biorad Gel Imager. This process is an Electrophoretic Mobility Shift Assay, which is further detailed in the Protocols tab of the Experiment section.</p>

<p style = "color: #000000; font-size: 16px;">The resulting gel image below depicts gel shifts for the positive control tiles PCAB, PC2A, and PC1A, along with control tiles (tiles with filler strands in place of aptamers). The shifts of the bands to higher equivalent base pair values for all three tiles shown when protein was added verify that the protein bound to the tiles and added weight to them as compared to the bands for lanes in which the tiles were not incubated with protein. These shifts are representative of all of the positive control tiles, as they all bound protein.</p>

<p style = "color: #000000; font-size: 16px;">These particular tiles also show an interesting phenomenon: tile PCAB does not run exactly like PC2A nor PC1A. Both PC2A and PC1A show brighter bands corresponding to the bands in the control lanes; meaning that a much lower percentage of PC2A and PC1A are bound to alpha-thrombin compared to PCAB. The major band of PCAB runs as one band as compared to the two shifted bands of PC2A. We hypothesize that this is because PC2A has two of the same aptamers incorporated into itself, which can each bind a separate protein molecule. In contrast, the form of PCAB bound only to one protein coordinated between the aptamers on the tile is likely much more stable than a form in which each aptamer is bound to a separate protein due to entropic effects. This also explains the dearth of unbound tiles at the control band site: since this complex is more stable, it has a lower KD and fewer tiles exist in an unbound form.</p><br>

<img src = "http://openwetware.org/images/a/a1/Screen_Shot_2014-10-23_at_4.11.42_AM.png"><br>

<p style = "color: #000000; font-size: 16px;">This gel is representative of gels run with randomized oligonucleotide inserts. The six lanes on the left between the ladders are different dilutions of the 6th and 7th lanes: the 2nd and 3rd lanes are 10x diluted, while the 4th and 5th lanes are 3x diluted. On the right side of the gel are positive and negative controls for binding alpha-thrombin.</p>

<p style = "color: #000000; font-size: 16px;">It is difficult to tell whether or not a small amount of the AT2R’ tiles in the protein-positive lanes have shifted. Optimisation is currently being done to try to recover any tiles that may be bound to protein but are too few in number to see with gel stain. This process involves cutting and eluting an area of the protein-positive lane above any visible signal, doing PCR for the randomized oligonucleotide insert strands, and comparing this to a control run with gel from the same area in the control lane. It is important to note that, though this procedure is susceptible to human error due to the difficulty of cutting the gel congruently in each lane, the overall outcome of the process can be tested in other ways. If the pool of randomized oligonucleotides is enriched, for example, assaying a new generation of tiles made with the enriched pool should show stronger shifts. </p>

<p style = "color: #000000; font-size: 16px;">The double nature of the bands in AT2R’ is characteristic of tiles of this nature: another previously-made tile, AT2R -- which incorporated two randomized oligonucleotides into the opposite sites as AT2R’ -- also showed such a signal in the control lanes. We suspect that this phenomenon is occurring due to some tiles failing to incorporate the aptamer strands. This should not be an issue, however, as the remaining tiles which have the inserts incorporated appear to be present in approximately equal amount, which should provide in itself a large enough selectable pool. Nonetheless, we are continuing to investigate the cause of the phenomenon and how it might possibly be prevented.</p><br>

<img src = "http://openwetware.org/images/5/56/Screen_Shot_2014-10-23_at_4.13.19_AM.png">