Biomod/2012/UTokyo/UT-Komaba/Experiment/Trioscillate System
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Concept
This system is quite similar to bistable system but it has three states, A, B and , each repressing the next one in a loop. Therefore it can never settle on any one of them and has to continuously. The diagram above shows the clockwise cycle of inhibition, the cycle of the states is A -> B -> C -> A -> B and it theoretically continues forever. However, we can design the system with the anticlockwise cycle of inhibition so that the state changes like A -> C -> B -> A -> C and it theoretically continues forever. Also, Tristate oscillator system does not need input, and it changes its state by itself. Therefore, when it is introduced to the DNA modified origami, we can make the DNA tablet which change its surface autonomously and cycle between three pictures. The purpose of the experiment is to find the best conditions of trioscillator system for continuous oscillations.
Experiment
October 2nd
To investigate the ideal condition of trioscillator system, we conducted experiments which checks three parts of this system. The cycle of the trioscillator canbe clockwise and anticlockwise so that we conducted the both experiment. All of the experiments below is the same cycle, X -> V -> Q. Inhmix contains V to inhX, X to inhQ and Q to inhV.
- The Experiment of XII
In the experiment, we first input DNA XII and templates CxII, V to inhV, X to inhQ and Q to inhV. After putting them in PCR for several hours, we put DNA VII so that we could observe the concentration of XII. The purpose of the experiment is to find out the best concentration of CxII. The best concentration enables us to decrease the number of XII.
- The Experiment of VII
This one is the same experiment of XII one. We first input DNA VII and templates CvII, V to inhV, X to inhQ and Q to inhV, and, after putting them in PCR for several hours, we put DNA QII so that we could observe the concentration of VII. The purpose of the experiment is to find out the best concentration of CvII. The best concentration enables us to decrease the number of VII.
- The Experiment of QII
This one is the same experiment of XII one. We first input DNA VII and templates CqII, V to inhV, X to inhQ and Q to inhV, and, after putting them in PCR for several hours, we put DNA XII so that we could observe the concentration of QII. The purpose of the experiment is to find out the best concentration of CqII. The best concentration enables us to decrease the number of QII.
October 11th
We tested the whole trioscillator system. To investigate the ideal condition, we conducted the experiment of both direction of inhibition (V-|X-|Q, V-|Q-|X) and searched the concentrations of CxII, CvII and CqII. We kept these tubes in 42°C for 10 hours.
- Inhibit Direction: V-|X-|Q
The solution contained DNA VII, XII and QII and templates CvII, CxII, CqII, V to inhX, X to inhQ and Q to inhV. However, the concentration of QII was ten times higher than that of VII or XII so that QII first inhibit VII. Then, there will be less inhX because of decreasing number of VII. Therefore, XII will make a lot of inhQ and the state will change from "there is only QII" to "there is only XII". As long as the reaction networks work well, the cycle of states may continues to change like "there is only QII" -> "XII" -> "VII" -> "QII"... The purpose of the experiment is to find out the best concentration of CxII and CqII.
- Inhibit Direction: V-|Q-|X
The solution contained DNA VII, XII and QII and templates CvII, CxII, CqII, V to inhX, X to inhQ and Q to inhV. However, the concentration of QII was ten times higher than that of VII or XII so that QII first inhibit XII. Then, there will be less inhX because of decreasing number of XII. Therefore, VII will make a lot of inhQ and the state will change from "there is only QII" to "there is only VII". As long as the reaction networks work well, the cycle of states may continues to change like "there is only QII" -> "XII" -> "VII" -> "QII"... The purpose of the experiment is to find out the best concentration of CvII and CqII.
In the experiment, we found out the good condition of the experiment of V-|Q-|X.
However, the result from that of V-|X-|Q was not accurate enough to introduce to the trioscillator system.
Therefore, we decided to do the experiment of V-|X-|Q again.
October 12th
- Inhibit Direction: V-|X-|Q
The purpose of the experiment is to find out the best condition for the experiment of inhibition V-|X-|Q. We change the concentration of CxII and CqII.
As the result, we found out that 0.50μL of CxII was too small, but 1,00μL of that was too big. We decided to search the best concentration of CxII between 0.50μL and 1.00μL.
October 18th
- Inhibit Direction: V-|X-|Q
According to the experiment in October 12th, [CxII] = 25nM is too low and [CxII] = 50nM is too high. Therefore, we investigated the best concentration of CxII.
October 19th
The purpose of this experiment is to find the best concentration of NBI because we got new NBI. We use trioscillator system and observe which tube shows the most radical change of the concentration.
As a result, lower concentration of NBI seems to be good. That is because, if there are a lot of NBI, inhibition works too much, and the reaction network does not oscillate.
