Team:MIT/ResultsProcessing

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<div class= "section" id="m2bio">
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<h1>Processing Overview</h1>
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We demonstrated the first strand displacement-based NOT gate, which will allow for more varied logic to be implemented using all forms of strand displacement, whether in vitro or in vivo, with RNA or DNA.
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We also introduced the hammerhead ribozyme, a powerful RNA-cutting tool, to iGEM and the parts registry.  We prove that our hammerhead ribozyme self-splices in a mammalian setting.  We intend to use the hammerhead ribozyme to manufacture RNA strand displacement gates in vivo.
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<h1>Not Gate in vitro</h1>
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<p><img src="https://static.igem.org/mediawiki/2012/9/9d/NOT_GATE2.png" width=600/>
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<br> <i>Figure 1 - DNA molecules that constitute the NOT GATE </i>
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The original strand displacement paper demonstrated AND and OR gates, but did not include NOT gates. We designed, built, and successfully tested a strand displacement NOT gate in vitro, expanding the computational structures possible with strand displacement.
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The design of our NOT gate is in Figure 1 above, where a letter with a '*' depicts a complementary domain to the one denoted by the letter alone. We arrived to this design after iterating through numerous other ideas, trying each time to reduce the number of molecules involved and their complexity.
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To understand the behavior of this NOT gate, it can be useful to consider two extreme cases: no input and saturation-level input.
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When the input is not present, molecule B can bind reversibly with A (by partially displacing a1) and reversibly with C (by partially displacing c2). When B binds with C, molecule D finishes the job and fully kicks c2 off of c.  c2 then triggers the readout E by irreversibly displacing e2 from e1.  Meanwhile, D also frees B from c1, making B catalytic and allowing it to react with more C molecules, amplifying the output.  Therefore we will see high fluorescence.
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When the input is present in high concentration, B binds to a2, partially displacing a2 from a1.  The input then binds to a1, completing what B started by fully and irreversibly separating a2 and a1.  This step was inspired by the mechanism of the cooperative hybridization (Cooperative Hybridization of Oligonucleotides,David Yu Zhang,JACS 2011).  Since B is stuck with a2, it can no longer displace c2 from C, and the readout pathway described above cannot continue. Consequently e2 cannot be displaced from the readout. Therefore we will see no fluorescence.
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The figure below shows experimental validation of our NOT gate design.  As predicted, the concentration of the output strand decreases as the concentration of the input strand increases.
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<img src="https://static.igem.org/mediawiki/2012/0/06/NOT_gate_small2.png" width=600/>
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<br> <i> Experimental verification of the in vitro NOT gate.  Output levels were measured through the displacement of a ROX-RQ strand, the e1 and e2 "readout" molecule in the header diagram.  As c2 displaces e2 from e1, the RQ quencher on e2 separates from the ROX fluorescent molecule on e1, and the ROX fluoresces.</i>
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One important consideration in implementing the NOT gate is the relative concentration of A with respect to input and B. If the concentration of A is too low, the cooperative hybridization between A, B, and a high concentration of input  can be slow.  In that case, B is free to displace c2 from C, triggering the output although the input level is high. On the other hand, if the concentration of A is too high, even without the presence of input, B will continuously reversibly bind with A.  Consequently, B is not available to displace c2 from C, and therefore we would not see a high level of output even when the level of input is low.
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In addition to the relative concentration of the different components, another important point is the absolute concentration of them.  This is mainly due to the equilibrium thermodynamics of cooperative hybridization. Since there are three reactants, but only two products, at low concentration the reactants are more favorable in the reaction, whereas at high concentration the products will be more favorable.
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In light of these considerations, we tuned the NOT gate first by finding a set of concentrations that give the correct qualitative behavior, and then by fine-adjusting A for the right trade-off between the interaction of A, input, and B when the input is high, and the interaction of A and B when the input is low.
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<h1>Not Gate modeling</h1>
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<p><img src="https://static.igem.org/mediawiki/2012/6/65/NOT_gate_in_vitro_vs_simulation_small.png" width=600/>
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<br> <i>Figure 2 - NOT GATE transfer function in vitro and by simulation using the software Visual DSD</i>
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The NOT GATE modeling was performed using Visual DSD, in Fig2 the overlay of the simulated transfer function and the in vitro one where the basal fluorescence this time was subtracted.
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<br> The simulation helped us to find the right trade off, as mentioned before, in the choice  of  the relative concentration of A with to respect of B and input.
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<br>For the rate constant we used the one from  the article "Scaling Up Digital Circuit Computation with DNA Strand Displacement Cascades, Lulu Qian  and Erik Winfree, Science, 2011".
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<div class= "section" id="m3bio">
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<h1>Hammerhead Ribozymes</h1>
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<img src= 'https://static.igem.org/mediawiki/2012/f/f2/Hammerheads.png' width=600/>
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<i><br> Key:Red: mKate, Blue: mKate-Hammerhead, Cyan: Hammerhead-mKate.
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<br>100,000 HEK293 cells were transfected with equimolar amounts of Hef1a:TagBFP, as a transfection marker and one of the following: Hef1a:mKate, Hef1a:Hammmerhead-mKate, and Hef1a:mKate-Hammerhead. 48 hrs later, cells were harvested and analyzed by flow cytometry. The histograms indicate that inserting a hammerhead ribozyme either before or after the mKate sequence prevents transcription of the fluorescent protein. However, this data does help deconvolute weather or not the hammerhead ribozymes prevent transcription by cleaving the mRNA, or occluding the ribosome.  </i>
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Latest revision as of 00:06, 27 October 2012

DEPRECATED. DO NOT EDIT.

Retrieved from "http://2012.igem.org/Team:MIT/ResultsProcessing"