Biomod/2012/UTokyo/UT-Komaba/Experiment/Normal Bistable: Difference between revisions

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[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_NormalBistableConcept.png|center]]
[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_NormalBistableConcept.png|center]]


In this Experiment,we tested basic bistable system, as explained in [[Biomod/2012/UTokyo/UT-Komaba/Idea#Bistable_System|Idea:Bistable System]].
In this Experiment,we tested the basic bistable system, as explained in [[Biomod/2012/UTokyo/UT-Komaba/Idea#Bistable_System|Idea section]].
The main purpose of the experiment is to find out the condition which changes the concentration of DNA most radically.
The main purpose of this experiment is to find out the condition which changes the concentration of DNA most radically.


==Method==
==Method==


===Investigating the Best Condition===
===Investigating the Best Conditions===
The actual experiment was far more complex than the simulation. That is because of the difference of reaction velocity in the reaction circuit, or unconsidered fact at the simulation. We conducted experiments in order to find out the best protocol for manipulating the bistable system.
The actual experiment was far more complex than the simulation. That is because of the difference of reaction velocity in the reaction circuit, not taken into account in the simulation. We conducted experiments in order to find out the best protocol for manipulating the bistable system.


===Monitoring Concentration===
===Monitoring Concentrations===
The bistable system itself does not provide the way of monitoring its state.
 
Therefore, we need real-time multiplexed monitoring system of DNA reaction circuits.
The bistable system itself does not provide a way to monitor its state.
Therefore, we need a real-time multiplexed monitoring system for DNA reaction circuits.
We used Quencher-free monitoring method [[Biomod/2012/UTokyo/UT-Komaba/Supplementary#References|[4]]] in our experiments.


==Experiment==
==Experiment==


===September 19th===
===September 14th===


We wanted to know the best concentration for the bistable system.
We did the first experiment of the bistable system. This time, we tried making the stable state and didn't try switching.
Two different DNA strands, "XII" and "VII", were used for the experiment.
There are four types of tubes, A, B, C and D.
We searched the best concentration of CvII and CxII separately.<br/>
A and B were with NBI, the nickase.
CvII is the template for doubling V, and CxII is the one of X.
A and C included VII at the beginning, while B and D did XII.
"X to inhV" is the template for making inhV DNA from X, and "V to inhX" is the one of V.
We change the concentration of CvII and CxII in order to find out the best concentration of them.
We keep PCR in 42°C.


*The Experiment of VII
The experiment was successfully done, and the opposite state of the initial one was observed in A and C.
Therefore, we could conclude that NBI worked properly on the situation.


(µL)
[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab_Notes#September_14th|More Information]]
{|
  !
  !Bst
  !NBI
  !tt-RecJ
  !DTT
  !BSA
  !Smix 4x
  !V to inhX
  !X to inhV
  !dTTP
  !VII
  !CvII
  !mQ
  !Total Amount
  |-
  |5x
  |0.21
  |0.84
  |0.32
  |0.21
  |0.21
  |5.25
  |0.42
  |0.42
  |0.21
  |0.11
  |0.11
  |12.69
  |21.00
  |-
  |10x
  |0.21
  |0.84
  |0.32
  |0.21
  |0.21
  |5.25
  |0.42
  |0.42
  |0.21
  |0.11
  |0.21
  |12.59
  |21.00
  |-
  |15x
  |0.21
  |0.84
  |0.32
  |0.21
  |0.21
  |5.25
  |0.42
  |0.42
  |0.21
  |0.11
  |0.32
  |12.48
  |21.00
  |-
  |20x
  |0.21
  |0.84
  |0.32
  |0.21
  |0.21
  |5.25
  |0.42
  |0.42
  |0.21
  |0.11
  |0.43
  |12.37
  |21.00
  |}




*The Experiment of XII
===September 19th===


(µL)
The concept of the experiment is to find out the best concentration of '''CvII''' which doubles the DNA '''VII''' and '''CxII''' which doubles the DNA '''XII'''.
{|
  !
  !Bst
  !NBI
  !tt-RecJ
  !DTT
  !BSA
  !Smix 4x
  !V to inhX
  !X to inhV
  !dTTP
  !XII
  !CxII
  !mQ
  !Total Amount
  |-
  |5x
  |0.21
  |0.84
  |0.32
  |0.21
  |0.21
  |5.25
  |0.42
  |0.42
  |0.21
  |0.11
  |0.11
  |12.69
  |21.00
  |-
  |10x
  |0.21
  |0.84
  |0.32
  |0.21
  |0.21
  |5.25
  |0.42
  |0.42
  |0.21
  |0.11
  |0.21
  |12.59
  |21.00
  |-
  |15x
  |0.21
  |0.84
  |0.32
  |0.21
  |0.21
  |5.25
  |0.42
  |0.42
  |0.21
  |0.11
  |0.32
  |12.48
  |21.00
  |-
  |20x
  |0.21
  |0.84
  |0.32
  |0.21
  |0.21
  |5.25
  |0.42
  |0.42
  |0.21
  |0.11
  |0.43
  |12.37
  |21.00
  |}


*Experiment for '''VII'''
[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_NormalBistableVII.png|center|The concept of the Experiment '''VII''']]


When several hours passed after keeping these tubes in 42°C, we injected XII 0.40µL in each VII tubes and VII 0.40µL in each XII tubes.
In this experiment, we first put DNA '''VII''', templates '''CvII''', templates '''V to inhX''' and templates '''X to inhV''' and kept them in a PCR machine at 42°C.
Then we kept these tubes in 42°C for a day.
Therefore, at the first step, the number of '''VII''' increased.
After the several hours since we put them in PCR, we put certain amount of '''XII''' in the tubes and find out which concentration of '''VII''' in the tube decrease most radically.
The purpose of the experiment is to find out the concentration of '''CvII''' which can decrease the number of '''VII''' most when we put '''XII''' but is still able to restart quickly when '''XII''' is finally degraded by the exonuclease.
This concentration of '''CvII''' should be used in the bistable system because it has the most capability to change the state from "only '''VII'''" to "only '''XII'''".


===September 24th===
*Experiment for '''XII'''
[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_NormalBistableXII.png|center|The concept of the Experiment '''XII''']]


We change the condition of the bistable experiment and put some tubes in PCR.
This experiment is the counterpart of the experiment for '''VII'''.
The concentration of Bst and X to inhV were different from the same experiment of September 19th.
The purpose of this experiment is to find out the concentration of '''CxII''' which can decrease the number of '''XII''' most when we put '''VII''' and is able start again efficiently.
We kept these tubes in 42°C.
This concentration of '''CxII''' can be used in the bistable system because it has the most capability of changing the state from "only '''XII'''" to "only '''VII'''".


*The Experiment of VII
The result could not be used for the bistable system because the inhibition of '''XII''' by '''VII''' was weak.
Therefore, we decided to do the same experiment in diffferent conditions.


(µL)
[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab Notes#September_19th|More Information]]
{| border="2"
  !
  !Bst
  !NBI
  !tt-RecJ
  !dTTP
  !BSA
  !Smix 4x
  !V to inhX
  !X to inhV
  !DTTP
  !VII
  !CvII
  !mQ
  !Total Amount
  |-
  |5x
  |0.17
  |0.84
  |0.32
  |0.21
  |0.21
  |5.25
  |0.42
  |0.32
  |0.21
  |0.11
  |0.11
  |12.83
  |21.00
  |-
  |10x
  |0.17
  |0.84
  |0.32
  |0.21
  |0.21
  |5.25
  |0.42
  |0.32
  |0.21
  |0.11
  |0.21
  |12.73
  |21.00
  |-
  |15x
  |0.17
  |0.84
  |0.32
  |0.21
  |0.21
  |5.25
  |0.42
  |0.32
  |0.21
  |0.11
  |0.32
  |12.62
  |21.00
  |-
  |20x
  |0.17
  |0.84
  |0.32
  |0.21
  |0.21
  |5.25
  |0.42
  |0.32
  |0.21
  |0.11
  |0.43
  |12.51
  |21.00
  |}




*The Experiment of XII
===September 24th===
 
(µL)
{|
  !
  !Bst
  !NBI
  !tt-RecJ
  !DTT
  !BSA
  !Smix 4x
  !V to inhX
  !X to inhV
  !dTTP
  !XII
  !CxII
  !mQ
  !Total Amount
  |-
  |5x
  |0.17
  |0.84
  |0.32
  |0.21
  |0.21
  |5.25
  |0.42
  |0.32
  |0.21
  |0.11
  |0.11
  |12.83
  |21.00
  |-
  |10x
  |0.17
  |0.84
  |0.32
  |0.21
  |0.21
  |5.25
  |0.42
  |0.32
  |0.21
  |0.11
  |0.21
  |12.73
  |21.00
  |-
  |15x
  |0.17
  |0.84
  |0.32
  |0.21
  |0.21
  |5.25
  |0.42
  |0.32
  |0.21
  |0.11
  |0.32
  |12.62
  |21.00
  |-
  |20x
  |0.17
  |0.84
  |0.32
  |0.21
  |0.21
  |5.25
  |0.42
  |0.32
  |0.21
  |0.11
  |0.43
  |12.51
  |21.00
  |}
 
 
When several hours had passed since we put them in PCR, we injected XII 0.40µL in each VII tubes and VII 0.40µL in each XII tubes.
Then we kept these tubes in 42°C for about 1 day as the bistable experiment we did before.
 
 
===October 2nd===
 
We lowered the concentratinons of D to V, and N to X.
Today, we conducted experiments in order to find out the ideal concentration of "X to inhV".
We kept these tubes in 42°C, and injected once each wells.
 
*The Experiment of XII
 
(µL)
{|
! BST !! NBI !! tt-RecJ !! DTT !! BSA !! Smix 4x !! V to inhX !! X to inhv !! dTTP !! XII !! CxII !! CvII !! mQ !! Total Amount
|-
| 0.17 || 0.84 || 0.32 || 0.21 || 0.21 || 5.25 || 0.42 || 0.21 || 0.21 || 0.11 || 0.11 || 0.42 || 12.54 || 21
|-
| 0.17 || 0.84 || 0.32 || 0.21 || 0.21 || 5.25 || 0.42 || 0.25 || 0.21 || 0.11 || 0.11 || 0.42 || 12.50 || 21
|-
| 0.17 || 0.84 || 0.32 || 0.21 || 0.21 || 5.25 || 0.42 || 0.28 || 0.21 || 0.11 || 0.11 || 0.42 || 12.47 || 21
|-
| 0.17 || 0.84 || 0.32 || 0.21 || 0.21 || 5.25 || 0.42 || 0.32 || 0.21 || 0.11 || 0.11 || 0.42 || 12.43 || 21
|}
injection: VII 0.6µL each


We conducted the same experiment as the one we did on September 19th.
We conducted the experiment of '''VII''' and that of '''XII'''.
The difference with the one nn September 19th is that the concentration of '''V to inhX''' is higher.
That is because the inhibition of '''XII''' by '''VII''' was a little weak so we could not get the good result on the 19th.
The purpose of the experiment is to find out the best concentrations for '''CvII''' and '''CxII'''.


*The Experiment of VII
[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab Notes#September_24th|More Information]]


(µL)
__NOEDITSECTION__
{|
! BST  !! NBI  !! tt-RecJ !! DTT !! BSA !! Smix 4x !! V to inhX !! X to inhv !! dTTP !! VII !! CxII !! CvII !! mQ !! Total Amount
|-
| 0.17 || 0.84 || 0.32 || 0.21 || 0.21 || 5.25 || 0.42 || 0.21 || 0.21 || 0.11 || 0.11 || 0.42 || 12.54 || 21
|-
| 0.17 || 0.84 || 0.32 || 0.21 || 0.21 || 5.25 || 0.42 || 0.25 || 0.21 || 0.11 || 0.11 || 0.42 || 12.50 || 21
|-
| 0.17 || 0.84 || 0.32 || 0.21 || 0.21 || 5.25 || 0.42 || 0.28 || 0.21 || 0.11 || 0.11 || 0.42 || 12.47 || 21
|-
| 0.17 || 0.84 || 0.32 || 0.21 || 0.21 || 5.25 || 0.42 || 0.32 || 0.21 || 0.11 || 0.11 || 0.42 || 12.43 || 21
|}
injection: XII 0.6µL each

Latest revision as of 02:19, 28 October 2012

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Concept

In this Experiment,we tested the basic bistable system, as explained in Idea section. The main purpose of this experiment is to find out the condition which changes the concentration of DNA most radically.

Method

Investigating the Best Conditions

The actual experiment was far more complex than the simulation. That is because of the difference of reaction velocity in the reaction circuit, not taken into account in the simulation. We conducted experiments in order to find out the best protocol for manipulating the bistable system.

Monitoring Concentrations

The bistable system itself does not provide a way to monitor its state. Therefore, we need a real-time multiplexed monitoring system for DNA reaction circuits. We used Quencher-free monitoring method [4] in our experiments.

Experiment

September 14th

We did the first experiment of the bistable system. This time, we tried making the stable state and didn't try switching. There are four types of tubes, A, B, C and D. A and B were with NBI, the nickase. A and C included VII at the beginning, while B and D did XII.

The experiment was successfully done, and the opposite state of the initial one was observed in A and C. Therefore, we could conclude that NBI worked properly on the situation.

More Information


September 19th

The concept of the experiment is to find out the best concentration of CvII which doubles the DNA VII and CxII which doubles the DNA XII.

  • Experiment for VII
The concept of the Experiment VII
The concept of the Experiment VII

In this experiment, we first put DNA VII, templates CvII, templates V to inhX and templates X to inhV and kept them in a PCR machine at 42°C. Therefore, at the first step, the number of VII increased. After the several hours since we put them in PCR, we put certain amount of XII in the tubes and find out which concentration of VII in the tube decrease most radically. The purpose of the experiment is to find out the concentration of CvII which can decrease the number of VII most when we put XII but is still able to restart quickly when XII is finally degraded by the exonuclease. This concentration of CvII should be used in the bistable system because it has the most capability to change the state from "only VII" to "only XII".

  • Experiment for XII
The concept of the Experiment XII
The concept of the Experiment XII

This experiment is the counterpart of the experiment for VII. The purpose of this experiment is to find out the concentration of CxII which can decrease the number of XII most when we put VII and is able start again efficiently. This concentration of CxII can be used in the bistable system because it has the most capability of changing the state from "only XII" to "only VII".

The result could not be used for the bistable system because the inhibition of XII by VII was weak. Therefore, we decided to do the same experiment in diffferent conditions.

More Information


September 24th

We conducted the same experiment as the one we did on September 19th. We conducted the experiment of VII and that of XII. The difference with the one nn September 19th is that the concentration of V to inhX is higher. That is because the inhibition of XII by VII was a little weak so we could not get the good result on the 19th. The purpose of the experiment is to find out the best concentrations for CvII and CxII.

More Information


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