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==Concept==
==Concept==


[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_TristableConcept.png|500px|center|The Concept of Trioscillate System]]
[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_TristableConcept.png|500px|center|The Concept of Trioscillator System]]
 
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 trioscillate system for continuous oscillations.


This system is quite similar to the bistable system but it has three states, A, B and C, each repressing the next one in the loop. Therefore it can never settle on any one of them and has to continuously update its own state.
The diagram above shows the clockwise cycle of inhibition. The system goes through states in the order A -> B -> C -> A -> B and it theoretically continues forever.
We can also design the system with an anticlockwise cycle of inhibition so that the state changes in the order A -> C -> B ->
A -> C and so on.
Also, the tristate oscillator system does not need input since it changes its state by itself.
Therefore, when it is used with the DNA modified origami, we can make the DNA tablet change its surface autonomously and cycle between three pictures.
The purpose of the experiment is to find the best conditions for the trioscillator system for continuous oscillations.


==Experiment==
==Experiment==
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===October 2nd===
===October 2nd===


To investigate the ideal condition of trioscillate system, we conducted experiments which checks three parts of this system.
To investigate the ideal conditions for the trioscillator system, we conducted experiments which check the three parts of this system.
The cycle of the trioscillate canbe clockwise and anticlockwise so that we conducted the both experiment.
The cycle of the trioscillator can be clockwise or anticlockwise so we conducted both experiments.
All of the experiments below is the same cycle, X -> V -> Q.
All of the experiments below are part of the same cycle, X -> V -> Q.
Inhmix contains V to inhX, X to inhQ and Q to inhV.
Inhmix contains V to inhX, X to inhQ and Q to inhV.


*The Experiment of XII
*XII Experiment
[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_TrioscillateSystemXII.png|center|The Concept of the Experiment of XII]]
[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_TrioscillateSystemXII.png|center|The Concept of 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.
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.
After storing them at 42°C for several hours, we add 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 purpose of the experiment is to find out the best concentration of CxII so that the system can change state quickly enough.
The best concentration enables us to decrease the number of XII.




*The Experiment of VII
*VII Experiment
[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_TrioscillateSystemVII.png|center|The Concept of the Experiment of VII]]
[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_TrioscillateSystemVII.png|center|The Concept of the Experiment of VII]]


This one is the same experiment of XII one.
This experiment is the same as the VII 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.
We first input DNA VII and templates CvII, V to inhV, X to inhQ and Q to inhV, and, after storing them 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 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
*QII Experiment
[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_TrioscillateSystemQII.png|center|The Concept of the Experiment of QII]]
[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_TrioscillateSystemQII.png|center|The Concept of the Experiment of QII]]


This one is the same experiment of XII one.
The last part of the system.
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.
We first input DNA VII and templates CqII, V to inhV, X to inhQ and Q to inhV, and, after storing them 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 purpose of the experiment is to find out the best concentration of CqII.
The best concentration enables us to decrease the number of QII.


[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab_Notes#October_2nd|More Information]]
[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab_Notes#October_2nd|More Information]]
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We tested the whole trioscillate 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 tested the whole trioscillator system. To investigate the ideal conditions, we conducted the experiment in both direction of inhibition (V-|X-|Q, V-|Q-|X) and searched the optimal concentrations of CxII, CvII and CqII.
We kept these tubes in 42°C for 10 hours.
We kept these tubes at 42°C for 10 hours.


*Inhibit Direction: V-|X-|Q
*Inhibit Direction: V-|X-|Q


[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_TrioscillateSystemAnticlockwise.png|center]]
[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_TrioscillateSystemAnticlockwise.png|center|Anticlockwise Cycle of the States]]


The solution contained DNA VII, XII and QII and templates CvII, CxII, CqII, V to inhX, X to inhQ and Q to inhV.
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.
However, the concentration of QII was ten times higher than that of VII or XII so that QII starts first and inhibits VII.
Then, there will be less inhX because of decreasing number of VII.
Then, there will be less inhX because of the 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".
Therefore, XII will make a lot of inhQ and the state will change from "QII" to "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"...
As long as the reaction network works well, the cycle of states may continue to change like "QII" -> "XII" -> "VII" -> "QII"...
The purpose of the experiment is to find out the best concentration of CxII and CqII.
The purpose of the experiment is to find out the best concentrations of CxII and CqII.




*Inhibit Direction: V-|Q-|X
*Inhibit Direction: V-|Q-|X


[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_TrioscillateSystemClockwise.png|center]]
[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_TrioscillateSystemClockwise.png|center|Clockwise Cycle of the States]]


The solution contained DNA VII, XII and QII and templates CvII, CxII, CqII, V to inhX, X to inhQ and Q to inhV.
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.
However, the concentration of QII was ten times higher than that of VII or XII so that QII starts first and inhibits XII.
Then, there will be less inhX because of decreasing number of XII.
Then, there will be less inhV because of the 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".
Therefore, VII will make a lot of inhQ and the state will change from "QII" to "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"...
As long as the reaction networks work well, the cycle of states may continues to change like "QII" -> "XII" -> "VII" -> "QII"...
The purpose of the experiment is to find out the best concentration of CvII and CqII.
The purpose of the experiment is to find out the best concentrations of CvII and CqII.




In the experiment, we found out the good condition of the experiment of V-|X-|Q.
In this experiment, we found out the good conditions for V-|Q-|X.
However, the result from that of V-|Q-|X was not accurate enough to introduce to the traioscillate system.
However, the results for V-|X-|Q was not accurate enough to use them in the trioscillator system.
Therefore, we decided to do the experiment of V-|Q-|X again.
Therefore, we decided to do the V-|X-|Q experiment again.


[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab_Notes#October_11th|More Information]]
[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab_Notes#October_11th|More Information]]
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===October 12th===
===October 12th===


We tested again the wells which worked well in before experiment.
*Inhibition Direction: V-|X-|Q


*Inhibit Direction: V-|X-|Q
The purpose of the experiment is to find out the best conditions for the inhibition cycle V-|X-|Q.
We made a ramp of concentrations of CxII and CqII.


(µL)
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 for the best concentration of CxII between 0.50μL and 1.00μL.
{|
! BST  !!  NBI  !!  tt-RecJ  !!  DTT  !!  BSA  !!  Smix 4x  !! inhmix !!  dTTP  !!  XII  !!  VII  !!  CxII  !!  CvII  !! CqII !!  mQ  !!  Total Amount
|-
| 0.16  || 0.80  || 0.30  || 0.20  || 0.20  || 5.00  || 0.40  || 0.10  || 0.11  || 0.11  || 0.50  || 0.40  || 0.50  || 11.22  || 20.00
|-
| 0.16  || 0.80  || 0.30  || 0.20  || 0.20  || 5.00  || 0.40  || 0.10  || 0.11  || 0.11  || 0.50  || 0.40  || 1.00  || 10.72  || 20.00
|-
| 0.16  || 0.80  || 0.30  || 0.20  || 0.20  || 5.00  || 0.40  || 0.10  || 0.11  || 0.11  || 0.50  || 0.40  || 1.50  || 10.22  || 20.00
|-
| 0.16  || 0.80  || 0.30  || 0.20  || 0.20  || 5.00  || 0.40  || 0.10  || 0.11  || 0.11  || 0.50  || 0.40  || 2.00  || 9.72  || 20.00
|-
| 0.16  || 0.80  || 0.30  || 0.20  || 0.20  || 5.00  || 0.40  || 0.10  || 0.11  || 0.11  || 1.00  || 0.40  || 0.50  || 10.72  || 20.00
|-
| 0.16  || 0.80  || 0.30  || 0.20  || 0.20  || 5.00  || 0.40  || 0.10  || 0.11  || 0.11  || 1.00  || 0.40  || 1.00  || 10.22  || 20.00
|-
| 0.16  || 0.80  || 0.30  || 0.20  || 0.20  || 5.00  || 0.40  || 0.10  || 0.11  || 0.11  || 1.00  || 0.40  || 1.50  || 9.72  || 20.00
|-
| 0.16  || 0.80  || 0.30  || 0.20  || 0.20  || 5.00  || 0.40  || 0.10  || 0.11  || 0.11  || 1.00  || 0.40  || 2.00  || 8.22  || 20.00
|}


[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab_Notes#October_12th|More Information]]
[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab_Notes#October_12th|More Information]]
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===October 18th===
===October 18th===


According to the 10/18 experiment, [CxII] = 25nM is too low and [CxII] = 50nM is too high. So today we investigated the best concentration of CxII.
*Inhibit Direction: V-|X-|Q
 
*Inhibit Direction: V-|Q-|X


(μL)
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.
! BST  !!  NBI  !!  tt-RecJ  !!  DTT  !!  BSA  !!  Smix 4x  !!  inhmix  !!  dTTP  !!  XII  !!  VII  !! QII !!  CxII !!  CvII  !!  CqII  !!  mQ  !!  Total Amount
|-
| 0.16  || 0.80  || 0.30  || 0.20  || 0.20  || 5.00  || 0.40  || 0.10  || 0.011  || 0.011  || 0.10  || 0.50  || 0.40  || 1.50  || 10.32  || 20.00
|-
| 0.16  || 0.80  || 0.30  || 0.20  || 0.20  || 5.00  || 0.40  || 0.10  || 0.011  || 0.011  || 0.10  || 0.65  || 0.40  || 1.50  || 10.17  || 20.00
|-
| 0.16  || 0.80  || 0.30  || 0.20  || 0.20  || 5.00  || 0.40  || 0.10  || 0.011  || 0.011  || 0.10  || 0.80  || 0.40  || 1.50  || 10.02  || 20.00
|-
| 0.16  || 0.80  || 0.30  || 0.20  || 0.20  || 5.00  || 0.40  || 0.10  || 0.011  || 0.011  || 0.10  || 0.95  || 0.40  || 1.50  || 9.87  || 20.00
|-
| 0.16  || 0.80  || 0.30  || 0.20  || 0.20  || 5.00  || 0.40  || 0.10  || 0.011  || 0.011  || 0.10  || 0.50  || 0.40  || 2.00  || 9.82  || 20.00
|-
| 0.16  || 0.80  || 0.30  || 0.20  || 0.20  || 5.00  || 0.40  || 0.10  || 0.011  || 0.011  || 0.10  || 0.65  || 0.40  || 2.00  || 9.67  || 20.00
|-
| 0.16  || 0.80  || 0.30  || 0.20  || 0.20  || 5.00  || 0.40  || 0.10  || 0.011  || 0.011  || 0.10  || 0.80  || 0.40  || 2.00  || 9.52  || 20.00
|-
| 0.16  || 0.80  || 0.30  || 0.20  || 0.20  || 5.00  || 0.40  || 0.10  || 0.011  || 0.011  || 0.10  || 0.95  || 0.40  || 2.00  || 9.37  || 20.00
|}


[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab_Notes#October_18th|More Information]]
[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab_Notes#October_18th|More Information]]


===October 19th===
===October 19th===


The purpose of this experiment is to find the best concentration of NBI because we got new NBI.
The purpose of this experiment is to find the best concentration of NBI, the nicking enzyme, because we got a new batch of NBI.
We use trioscillate system and observe which tube shows the most radical change of the concentration.
We use the trioscillator system and observe which tube behaves the best.


(µL)
As a result, lower concentration of NBI seemed to be good.  
{|
That is because, if there are a lot of NBI, too much inhibitors are produced, and the reaction network does not oscillate.
! BST  !!  NBI !!  tt-RecJ  !!  DTT  !!  BSA  !!  Smix 4x  !!  inhmix  !!  dTTP  !!  XII  !!  VII  !!  QII  !!  CxII  !!  CvII  !!  CqII  !!  mQ  !!  Total Amount
|-
| 0.080  || 0.025  || 0.15  || 0.10  || 0.10  || 2.50  || 0.20  || 0.050  || 0.0056  || 0.0056  || 0.050  || 0.075  || 0.20  || 0.075  || 6.38  || 10.000
|-
| 0.080  || 0.050  || 0.15  || 0.10  || 0.10  || 2.50  || 0.20  || 0.050  || 0.0056  || 0.0056  || 0.050  || 0.075  || 0.20  || 0.075  || 6.36  || 10.000
|-
| 0.080  || 0.075  || 0.15  || 0.10  || 0.10  || 2.50  || 0.20  || 0.050  || 0.0056  || 0.0056  || 0.050  || 0.075  || 0.20  || 0.075  || 6.33  || 10.000
|-
| 0.080  || 0.100  || 0.15  || 0.10  || 0.10  || 2.50  || 0.20  || 0.050  || 0.0056  || 0.0056  || 0.050  || 0.075  || 0.20  || 0.075  || 6.31  || 10.000
|-
| 0.080  || 0.125  || 0.15  || 0.10  || 0.10  || 2.50  || 0.20  || 0.050  || 0.0056  || 0.0056  || 0.050  || 0.075  || 0.20  || 0.075  || 6.28  || 10.000
|-
| 0.080  || 0.150  || 0.15  || 0.10  || 0.10  || 2.50  || 0.20  || 0.050  || 0.0056  || 0.0056  || 0.050  || 0.075  || 0.20  || 0.075  || 6.26  || 10.000
|-
| 0.080  || 0.175  || 0.15  || 0.10  || 0.10  || 2.50  || 0.20  || 0.050  || 0.0056  || 0.0056  || 0.050  || 0.075  || 0.20  || 0.075  || 6.23  || 10.000
|-
| 0.080  || 0.200  || 0.15  || 0.10  || 0.10  || 2.50  || 0.20  || 0.050  || 0.0056  || 0.0056  || 0.050  || 0.075  || 0.20  || 0.075  || 6.21  || 10.000
|-
| 0.080  || 0.225  || 0.15  || 0.10  || 0.10  || 2.50  || 0.20  || 0.050  || 0.0056  || 0.0056  || 0.050  || 0.075  || 0.20  || 0.075  || 6.18  || 10.000
|-
| 0.080  || 0.250  || 0.15  || 0.10  || 0.10  || 2.50  || 0.20  || 0.050  || 0.0056  || 0.0056  || 0.050  || 0.075  || 0.20  || 0.075  || 6.16  || 10.000
|}


According to the result, lower concentration of NBI seems to be good.
[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab_Notes#October_19th|More Information]]


[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab_Notes#October_19th|More Information]]
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Concept

The Concept of Trioscillator System
The Concept of Trioscillator System

This system is quite similar to the bistable system but it has three states, A, B and C, each repressing the next one in the loop. Therefore it can never settle on any one of them and has to continuously update its own state. The diagram above shows the clockwise cycle of inhibition. The system goes through states in the order A -> B -> C -> A -> B and it theoretically continues forever. We can also design the system with an anticlockwise cycle of inhibition so that the state changes in the order A -> C -> B -> A -> C and so on. Also, the tristate oscillator system does not need input since it changes its state by itself. Therefore, when it is used with the DNA modified origami, we can make the DNA tablet change its surface autonomously and cycle between three pictures. The purpose of the experiment is to find the best conditions for the trioscillator system for continuous oscillations.

Experiment

October 2nd

To investigate the ideal conditions for the trioscillator system, we conducted experiments which check the three parts of this system. The cycle of the trioscillator can be clockwise or anticlockwise so we conducted both experiments. All of the experiments below are part of the same cycle, X -> V -> Q. Inhmix contains V to inhX, X to inhQ and Q to inhV.

  • XII Experiment
The Concept of the Experiment of XII
The Concept of 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 storing them at 42°C for several hours, we add 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 so that the system can change state quickly enough.


  • VII Experiment
The Concept of the Experiment of VII
The Concept of the Experiment of VII

This experiment is the same as the VII one. We first input DNA VII and templates CvII, V to inhV, X to inhQ and Q to inhV, and, after storing them 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.


  • QII Experiment
The Concept of the Experiment of QII
The Concept of the Experiment of QII

The last part of the system. We first input DNA VII and templates CqII, V to inhV, X to inhQ and Q to inhV, and, after storing them 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.

More Information


October 11th

We tested the whole trioscillator system. To investigate the ideal conditions, we conducted the experiment in both direction of inhibition (V-|X-|Q, V-|Q-|X) and searched the optimal concentrations of CxII, CvII and CqII. We kept these tubes at 42°C for 10 hours.

  • Inhibit Direction: V-|X-|Q
Anticlockwise Cycle of the States
Anticlockwise Cycle of the States

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 starts first and inhibits VII. Then, there will be less inhX because of the decreasing number of VII. Therefore, XII will make a lot of inhQ and the state will change from "QII" to "XII". As long as the reaction network works well, the cycle of states may continue to change like "QII" -> "XII" -> "VII" -> "QII"... The purpose of the experiment is to find out the best concentrations of CxII and CqII.


  • Inhibit Direction: V-|Q-|X
Clockwise Cycle of the States
Clockwise Cycle of the States

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 starts first and inhibits XII. Then, there will be less inhV because of the decreasing number of XII. Therefore, VII will make a lot of inhQ and the state will change from "QII" to "VII". As long as the reaction networks work well, the cycle of states may continues to change like "QII" -> "XII" -> "VII" -> "QII"... The purpose of the experiment is to find out the best concentrations of CvII and CqII.


In this experiment, we found out the good conditions for V-|Q-|X. However, the results for V-|X-|Q was not accurate enough to use them in the trioscillator system. Therefore, we decided to do the V-|X-|Q experiment again.

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October 12th

  • Inhibition Direction: V-|X-|Q

The purpose of the experiment is to find out the best conditions for the inhibition cycle V-|X-|Q. We made a ramp of concentrations 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 for the best concentration of CxII between 0.50μL and 1.00μL.

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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.

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October 19th

The purpose of this experiment is to find the best concentration of NBI, the nicking enzyme, because we got a new batch of NBI. We use the trioscillator system and observe which tube behaves the best.

As a result, lower concentration of NBI seemed to be good. That is because, if there are a lot of NBI, too much inhibitors are produced, and the reaction network does not oscillate.

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