Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Gel Verification: Difference between revisions
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{| class="wikitable" border="1 | {| class="wikitable" border="1" | ||
|- | |- | ||
| Lane # | | Lane # | ||
Line 67: | Line 67: | ||
| Information from the gel | | Information from the gel | ||
|- | |- | ||
| Lane 1 | W | Control | | Lane 1 | ||
| W | |||
| Control | |||
|- | |- | ||
| Lane 2 | | Lane 2 | ||
Line 119: | Line 121: | ||
Lane 1 | {| class="wikitable" border="1" | ||
Lane 2 | |- | ||
Lane 3 | | Lane # | ||
Lane 4 | | Input and Expected Reaction | ||
Lane 5 | | Information from the gel | ||
Lane 6 | |- | ||
Lane 7 | | Lane 1 | ||
Lane 8 | | W | ||
Lane 9 | | Control | ||
Lane 10 | |- | ||
Lane 11 | | Lane 2 | ||
Lane 12 | | TR1 | ||
Lane 13 | | Control | ||
Lane 14 | |- | ||
| Lane 3 | |||
| TR2 | |||
| Control | |||
|- | |||
| Lane 4 | |||
| PTR2 | |||
| Control | |||
|- | |||
| Lane 5 | |||
| TR2 + PTR2 → (TR2-PTR2) | |||
| Track 2 binds with its probe. This will allow us to distinguish between Track 2 and Track 1 in future experiments. <br /> | |||
|- | |||
| Lane 6 | |||
| W + (TR2-PTR2) → W-TR2-PTR2 | |||
| Track 2's probe does not interfere with the binding of the Walker to track 2 | |||
|- | |||
| Lane 7 | |||
| (W-TR1) | |||
| Control | |||
|- | |||
| Lane 8 | |||
| (W-TR1) + (TR2-PTR2) ↔ (W-TR2-PTR2) + TR1 | |||
| Walker can move between tracks, specifically from Track 1 to Track 2. The system should equalize with walkers on both tracks. | |||
|- | |||
| Lane 9 | |||
| (W-TR2-PTR2) + TR1 ↔ (W-TR1) + (TR2-PTR2) | |||
| Walker can move between tracks, specifically from Track 2 to Track 1. The system should equalize with walkers on both tracks | |||
|- | |||
| Lane 10 | |||
| (WI-WT) | |||
| Control | |||
|- | |||
| Lane 11 | |||
| (W-WI) | |||
| Control | |||
|- | |||
| Lane 12 | |||
| (W-TR1-WI) | |||
| Control | |||
|- | |||
| Lane 13 | |||
| (W-TR1-WI) + (TR2-PTR2) | |||
| NULL experiment - Walker Inhibitor does in fact inhibit walking | |||
|- | |||
| Lane 14 | |||
| (W-TR1-WI) + (TR2-PTR2) + WT → (WI-WT) + TR1 + (W-TR2-PTR2) + (W-TR1-WI) +(TR2-PTR2) | |||
| Walker Trigger initiates walking | |||
|} | |||
*In lanes 12, 13, and 14 there is 2x of WI, and in lane 14 there is 4x of WT. | *In lanes 12, 13, and 14 there is 2x of WI, and in lane 14 there is 4x of WT. | ||
Line 143: | Line 194: | ||
[[Image:equilibrium.jpg|600px|center|thumb|NUPACK simulation; equilibrium between (walker – track 1) and (walker – track2) after random walking in solution ]]<br /> | [[Image:equilibrium.jpg|600px|center|thumb|NUPACK simulation; equilibrium between (walker – track 1) and (walker – track2) after random walking in solution ]]<br /> | ||
==Gel 3== | ==Gel 3== | ||
Line 152: | Line 202: | ||
[[Image:Dnawesome_gel3.png|600px|thumb|center]]<br /> | [[Image:Dnawesome_gel3.png|600px|thumb|center]]<br /> | ||
{| class="wikitable" border="1" | |||
Lane 1 | |- | ||
Lane 2 | | Lane # | ||
Lane 3 | | Input and Expected Reaction | ||
Lane 4 | | Information from the gel | ||
Lane 5 | |- | ||
Lane 6 | | Lane 1 | ||
| CG1 | |||
| Control | |||
|- | |||
| Lane 2 | |||
| CGI | |||
| Control | |||
|- | |||
| Lane 3 | |||
| CGT | |||
| Control | |||
|- | |||
| Lane 4 | |||
| CG1 + CGI → (CG1-CGI) | |||
| Cargo goal inhibitor binds to the cargo goal. | |||
|- | |||
| Lane 5 | |||
| CGI + CGT →(CGI-CGT) | |||
| Cargo goal trigger binds with the cargo goal inhibitor | |||
|- | |||
| Lane 6 | |||
| (CG1-CGI) + CGT → (CGI-CGT)+ CG1 | |||
| Cargo goal trigger is capable of stripping the Cargo goal inhibitor off of the Cargo goal <br | |||
|} | |||
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[[Image:Dnawesome_gel4.png|600px|thumb|center]]<br /> | [[Image:Dnawesome_gel4.png|600px|thumb|center]]<br /> | ||
{| class="wikitable" border="1" | |||
Lane 1 | |- | ||
Lane 2 | | Lane # | ||
Lane 3 | | Input and Expected Reaction | ||
Lane 4 | | Information from the gel | ||
Lane 5 | |- | ||
Lane 6 | | Lane 1 | ||
Lane 7 | | C1, TR1 | ||
Lane 8 | | Control<br /> | ||
Lane 9 | |- | ||
Lane 10 | | Lane 2 | ||
Lane 11 | | TR2 | ||
Lane 12 | | Control <br /> | ||
Lane 13 | |- | ||
Lane 14 | | Lane 3 | ||
| W, CA | |||
| Control<br /> | |||
|- | |||
| Lane 4 | |||
| C1 + CA →(C1-CA) | |||
| Cargo binds to the Cargo Attaching strand <br /> | |||
|- | |||
| Lane 5 | |||
| W + C1 → (W-C1) | |||
| Cargo binds to the Walker <br /> | |||
|- | |||
| Lane 6 | |||
| W + (C1-CA) →(W-C1) + CA | |||
| Walker picks up the Cargo by stripping off of the Cargo Attaching strand<br /> | |||
|- | |||
| Lane 7 | |||
| (W-TR1) | |||
| Control<br /> | |||
|- | |||
| Lane 8 | |||
| (W-TR2) | |||
| Control<br /> | |||
|- | |||
| Lane 9 | |||
| W + TR1 + C1 →(W-TR1-C1) | |||
| Track 1 does not interfere with the binding between the Walker and Cargo.<br /> | |||
|- | |||
| Lane 10 | |||
| W + TR2 + C1 →(W-TR2-C1) | |||
| Track 2 does not interfere with the binding between the Walker and Cargo.<br /> | |||
|- | |||
| Lane 11 | |||
| (W-TR1) + (C1-CA) →(W-TR1-C1) + CA | |||
| Track 1 does not interfere with the Walker picking up the Cargo off of the Cargo Attaching strand. <br /> | |||
|- | |||
| Lane 12 | |||
|(W-TR2) + (C1-CA) →(W-TR2-C1) + CA | |||
| Track 2 does not interfere with the Walker picking up the Cargo off of the Cargo Attaching strand. <br /> | |||
|- | |||
| Lane 13 | |||
| (W-TR1-C1) + CA | |||
| NULL experiment. Picking up mechanism is irreversible <br /> | |||
|- | |||
| Lane 14 | |||
| (W-TR2-C1) + CA | |||
| NULL experiment. Picking up mechanism is irreversible<br /> | |||
|} | |||
==Gel 5== | ==Gel 5== | ||
Line 192: | Line 311: | ||
[[Image:Dnawesome_gel5.png|600px|thumb|center]]<br /> | [[Image:Dnawesome_gel5.png|600px|thumb|center]]<br /> | ||
{| class="wikitable" border="1" | |||
Lane 1 | |- | ||
Lane 2 | | Lane # | ||
Lane 3 | | Input and Expected Reaction | ||
Lane 4 | | Information from the gel | ||
Lane 5 | |- | ||
Lane 6 | | Lane 1 | ||
Lane 7 | | C1 | ||
Lane 8 | | Control<br /> | ||
Lane 9 | |- | ||
Lane 10 | | Lane 2 | ||
Lane 11 | | TR1 | ||
Lane 12 | | Control <br /> | ||
|- | |||
| Lane 3 | |||
| (TR2-PTR2) | |||
| Control <br /> | |||
|- | |||
| Lane 4 | |||
| (W-TR1-C1) | |||
| Control <br /> | |||
|- | |||
| Lane 5 | |||
| W + TR2 + PTR2 + C1 → (W-TR2-PTR2-C1) | |||
| Control <br /> | |||
|- | |||
| Lane 6 | |||
| (W-TR1-C1) + (TR2-PTR2) ↔ (W-TR2-PTR2-C1) + TR1 | |||
| Walker walking while carrying a cargo. Reached equilibrium <br /> | |||
|- | |||
| Lane 7 | |||
| (W-TR2-PTR2-C1) + TR1 ↔ (W-TR1-C1) +(TR2-PTR2) | |||
| Walker walking while carrying a cargo. Reached equilibrium <br /> | |||
|- | |||
| Lane 8 | |||
| (W-C1) | |||
| Control<br /> | |||
|- | |||
| Lane 9 | |||
| (W-TR1) | |||
| Control<br /> | |||
|- | |||
| Lane 10 | |||
| (W-TR2) | |||
| Control <br /> | |||
|- | |||
| Lane 11 | |||
| (W-TR2-C1) | |||
| Control <br /> | |||
|- | |||
| Lane 12 | |||
| (W-TR2-PTR2) | |||
| Control <br /> | |||
|} | |||
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[[Image:Dnawesome_gel6.png|600px|thumb|center]]<br /> | [[Image:Dnawesome_gel6.png|600px|thumb|center]]<br /> | ||
{| class="wikitable" border="1" | |||
Lane 1 | |- | ||
Lane 2 | | Lane # | ||
Lane 3 | | Input and Expected Reaction | ||
Lane 4 | | Information from the gel | ||
Lane 5 | |- | ||
Lane 6 | | Lane 1 | ||
Lane 7 | | W | ||
Lane 8 | | Control<br /> | ||
Lane 9 | |- | ||
Lane 10 | | Lane 2 | ||
Lane 11 | | CG1 | ||
Lane 12 | | Control<br /> | ||
Lane 13 | |- | ||
Lane 14 | | Lane 3 | ||
| PCG1 | |||
| Control <br /> | |||
|- | |||
| Lane 4 | |||
| CG1 + PCG1 → (CG1-PCG1) | |||
| cargo goal binds to the probe for the cargo goal <br /> | |||
|- | |||
| Lane 5 | |||
| C1 + CG1 + PCG1 → (W-C1-PCG1) | |||
| cargo binds to the cargo goal with the probe<br /> | |||
|- | |||
| Lane 6 | |||
| (W-C1) | |||
| Control <br /> | |||
|- | |||
| Lane 7 | |||
| (W-C1) + (CG1-PCG1) ↔ (C1-CG1-PCG1) + W | |||
| Dropping off mechanism <br /> | |||
|- | |||
| Lane 8 | |||
| W + (C1-CG1-PCG1) | |||
| NULL experiment : Dropping off mechanism is irreversible <br /> | |||
|- | |||
| Lane 9 | |||
| (W-TR1) | |||
| Control <br /> | |||
|- | |||
| Lane 10 | |||
| (W-TR1-C1) | |||
| Control<br /> | |||
|- | |||
| Lane 11 | |||
| (W-TR1-C1) + (CG1-PCG1) ↔ (C1-CG1-PCG1) +(W-TR1) | |||
| Dropping off mechanism with track 1 <br /> | |||
|- | |||
| Lane 12 | |||
| (W-TR2) | |||
| Control<br /> | |||
|- | |||
| Lane 13 | |||
| (W-TR2-C1) | |||
| Control <br /> | |||
|- | |||
| Lane 14 | |||
| (W-TR2-C1) + (CG1-PCG1) ↔ (C1-CG1-PCG1) + (W-TR2) | |||
| Dropping off mechanism with track 2 <br /> | |||
|} | |||
==Gel 7== | ==Gel 7== | ||
Line 238: | Line 444: | ||
[[Image:dnawesome_gel7.png|600px|thumb|center]]<br /> | [[Image:dnawesome_gel7.png|600px|thumb|center]]<br /> | ||
{| class="wikitable" border="1" | |||
|- | |||
| Lane # | |||
| Input and Expected Reaction | |||
| Information from the gel | |||
|- | |||
| Lane 1 | |||
| W | |||
| Control<br /> | |||
|- | |||
| Lane 2 | |||
| TR1 | |||
| Control<br /> | |||
|- | |||
| Lane 3 | |||
| TR2 | |||
| Control <br /> | |||
|- | |||
| Lane 4 | |||
| WG | |||
| Control<br /> | |||
|- | |||
| Lane 5 | |||
| PWG | |||
| Control<br /> | |||
|- | |||
| Lane 6 | |||
| (W-TR1) | |||
| Control <br /> | |||
|- | |||
| Lane 7 | |||
| (W-TR2) | |||
| Control <br /> | |||
|- | |||
| Lane 8 | |||
| W+WG → (W-WG) | |||
| Walker binds to a Walker goal. <br /> | |||
|- | |||
| Lane 9 | |||
| WG + PWG → (WG-PWG) | |||
| Walker goal binds to the probe. Works as a control here. <br /> | |||
|- | |||
| Lane 10 | |||
| W+WG + PWG → (W-WG-PWG) | |||
| Control | |||
|- | |||
| Lane 11 | |||
| (W-TR1) + (WG-PWG) → (W-WG-PWG) + TR1 | |||
| Walker walks from track 1 to a Walker goal.<br /> | |||
|- | |||
| Lane 12 | |||
| (W-TR2) + (WG-PWG) → (W-WG-PWG) + TR2 | |||
| Walker walks from track 2 to a Walker goal. <br /> | |||
|- | |||
| Lane 13 | |||
| (W-WG-PWG) + TR1 → NULL | |||
| Walker goal is irreversible <br /> | |||
|- | |||
| Lane 14 | |||
|(W-WG-PWG) + TR2 → NULL | |||
| walker goal is irreversible <br /> | |||
|} | |||
{{Template:DeoxyriboNucleicAwesomeFooter}} | {{Template:DeoxyriboNucleicAwesomeFooter}} |
Latest revision as of 09:26, 3 November 2011
Wednesday, May 15, 2024
|
Gel VerificationAll of the essential mechanisms in our system were verified in solution using polyacrylamide gel electrophoresis. These mechanisms include: walker-track binding, triggering the walker, walking from one track to another, picking up cargo, walking while carrying cargo, triggering the cargo goal, dropping off cargo, and irreversibly walking from tracks to the walker goal. Each gel has several control lanes (marked in green), where control lanes are either single strands, or previously verified combinations of strands. Positive controls are marked with (+) below the lane number, and negative controls are marked with (-) below the lane number. We define a negative control as one that should not appear in any reaction in that gel, even though some may in practice appear due to stoichiometric errors etc. Lanes that test the binding of certain strands are labeled in blue, whereas lanes that test strand displacement reactions are labeled in red. Null experiments (ones where we expect no reaction to occur, but provide comparisons for reactions where we expect the end result to be the same or similar) are labeled in black. All inputs in a lane were intended to be equimolar unless otherwise noted. Probes were used when it seemed necessary to distinguish strands or complexes of similar length (for example PTR2 was used with track 2 to distinguish it from track 1, although we found that TR1 always appears slightly higher in the gel than TR2, and likewise when they are bound to the walker, perhaps due to some minor secondary structure). While the mechanisms all appear to behave properly in solution, there were a few mysteries, which may or may not matter as the mechanisms are translated onto origami. One such mystery is the apparent absence of TR2-PTR2 in lanes 6 and 7 of gel 4. There a couple possibilities for this. One is that it is just very low intensity, and since it should be close to the bands for W-TR1-C1, it blends in. Another is that there were stoichiometric issues, so almost all of the TR2 is in W-TR2-PTR2-C1. In any case, it does not seem possible for it to not exist if stoichiometry was correct, given the results in those two lanes. Another mystery is that in lanes 7 and 8 of gel 6 the bands that correspond to the walker appear somewhat higher than expected. No logical explanation can be found for this, and it seems ignorable, except for the fact that in a similar gel using old cargo and cargo goal strands, the same effect appeared! Finally, in lane 4 of gel 7, the walker goal appears much higher than a 47 nucleotide strand should appear (compare to the tracks). This was also seen in other gels, which suggests there is probably some polymerization of the strand (or otherwise some secondary structure), but since the rest of the gel suggests the walker goal behaves as expected, it will be ignored.
Gel 1Ability of the walker to bind to its tracks and the walker triggering mechanism.
Gel 2Random walking mechanism and initiation of walking by triggering the walker
Gel 3Cargo goal triggering mechanism
Gel 4Picking-up mechanism
Gel 5Walker walking while carrying a cargo
Gel 6Dropping off mechanism
Gel 7Termination of random walking by reaching the walker goal
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