Biomod/2012/UTokyo/UT-Komaba/Experiment/Normal Bistable
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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.
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
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
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.
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.
