Biomod/2012/UTokyo/UT-Komaba/Experiment/Normal Bistable
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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 14th
Bistable System
(µL)
| Smix 4x | DTT | BSA | CvII | CxII | XtoinhV | VtoinhX | tt-Recj | BST | NBI | mQ | VII | XII | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Group1 | 5 | 0.25 | 0.25 | 0.375 | 0.125 | 0.5 | 0.5 | 0.375 | 0.2 | 0.25 | 0.25 | 0.5 | |
| Group2 | 5 | 0.25 | 0.25 | 0.375 | 0.125 | 0.5 | 0.5 | 0.375 | 0.2 | 0.25 | 0.25 | 0.5 | |
| Groiup3 | 5 | 0.25 | 0.25 | 0.375 | 0.125 | 0.5 | 0.5 | 0.375 | 0.2 | 0.5 | 0.5 | ||
| Group4 | 5 | 0.25 | 0.25 | 0.375 | 0.125 | 0.5 | 0.5 | 0.375 | 0.2 | 0.5 | 0.5 |
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
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
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".
The result could not be introduced to the bistable system because the inhibition of XII to VII was weak. Therefore, we decided to do the same experiment in diffferent condition.
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 XII to VII 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.
