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

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If you want more detailed information, please read [[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab Notes#September_19th|Lab notes]]
If you want more detailed information, please read [[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab Notes#September_19th|Lab notes]]


===September 24th===
===September 24th===


We change the condition of the bistable experiment and put some tubes in PCR.
We conducted the same experiment as the one we did in September 19th.
The concentration of Bst and X to inhV were different from the same experiment of September 19th.
We conducted the experiment of VII and that of XII.
We kept these tubes in 42°C.
The different point from the one in September 19 th is that the concentration of V to inhX is higher.
 
That is because the inhibition of X to C was a little weak so  that we could not get the good result in 19th.
*The Experiment of VII
The purpose of the experiment is to find out the best concentration of CvII and CxII.
 
(µL)
{| 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
 
(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.




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Concept

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

Method

Investigating the Best Condition

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.

Monitoring Concentration

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.

Experiment

September 19th

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

  • The Experiment of 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 PCR in 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. This concentration of CvII can be introduced to the bistable system because they have the most capability of changing the state from "there is only VII" to "there is only XII".

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

This experiment is the completely opposite one of the experiment of VII. The purpose of the experiment is to find out the concentration of CxII which can decrease the number of XII most when we put VII. This concentration of CxII can be introduced to the bistable system because they have the most capability of changing the state from "there is only XII" to "there is only VII".

If you want more detailed information, please read Lab notes


September 24th

We conducted the same experiment as the one we did in September 19th. We conducted the experiment of VII and that of XII. The different point from the one in September 19 th is that the concentration of V to inhX is higher. That is because the inhibition of X to C was a little weak so that we could not get the good result in 19th. The purpose of the experiment is to find out the best concentration of CvII and CxII.


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


  • The Experiment of VII

(µL)

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

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