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

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===Monitoring Concentration===
===Monitoring Concentration===
The bistable system itself does not provide the way of monitoring its state.
The bistable system itself does not provide the way of monitoring its state.
Therefore, we needed real-time multiplexed monitoring system of DNA reaction circuits.
Therefore, we need real-time multiplexed monitoring system of DNA reaction circuits.


==Experiment==
==Experiment==

Revision as of 09:11, 22 October 2012

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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

We wanted to know the best concentration for the bistable system. Two different DNA strands, "XII" and "VII", were used for the experiment. We searched the best concentration of CvII and CxII separately.
CvII is the template for doubling V, and CxII is the one of X. "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

(µL)

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

(µL)

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


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. Then we kept these tubes in 42°C for a day.


September 24th

We change the condition of the bistable experiment and put some tubes in PCR. The concentration of Bst and X to inhV were different from the same experiment of September 19th. We kept these tubes in 42°C.

  • The Experiment of VII

(µL)

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.


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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