# Biomod/2012/UTokyo/UT-Komaba/Experiment/Trioscillate System

## Concept

This system is quite similar to the bistable system but it has three states, A, B and C, each repressing the next one in the loop. Therefore it can never settle on any one of them and has to continuously update its own state. The diagram above shows the clockwise cycle of inhibition. The system goes through states in the order A -> B -> C -> A -> B and it theoretically continues forever. We can also design the system with an anticlockwise cycle of inhibition so that the state changes in the order A -> C -> B -> A -> C and so on. Also, the tristate oscillator system does not need input since it changes its state by itself. Therefore, when it is used with the DNA modified origami, we can make the DNA tablet change its surface autonomously and cycle between three pictures. The purpose of the experiment is to find the best conditions for the trioscillator system for continuous oscillations.

## Experiment

### October 2nd

To investigate the ideal conditions for the trioscillator system, we conducted experiments which check the three parts of this system. The cycle of the trioscillator can be clockwise or anticlockwise so we conducted both experiments. All of the experiments below are part of the same cycle, X -> V -> Q. Inhmix contains V to inhX, X to inhQ and Q to inhV.

• XII Experiment

In the experiment, we first input DNA XII and templates CxII, V to inhV, X to inhQ and Q to inhV. After storing them at 42°C for several hours, we add DNA VII so that we could observe the concentration of XII. The purpose of the experiment is to find out the best concentration of CxII so that the system can change state quickly enough.

• VII Experiment

This experiment is the same as the VII one. We first input DNA VII and templates CvII, V to inhV, X to inhQ and Q to inhV, and, after storing them for several hours, we put DNA QII so that we could observe the concentration of VII. The purpose of the experiment is to find out the best concentration of CvII.

• QII Experiment

The last part of the system. We first input DNA VII and templates CqII, V to inhV, X to inhQ and Q to inhV, and, after storing them for several hours, we put DNA XII so that we could observe the concentration of QII. The purpose of the experiment is to find out the best concentration of CqII.

### October 11th

We tested the whole trioscillator system. To investigate the ideal conditions, we conducted the experiment in both direction of inhibition (V-|X-|Q, V-|Q-|X) and searched the optimal concentrations of CxII, CvII and CqII. We kept these tubes at 42°C for 10 hours.

• Inhibit Direction: V-|X-|Q

The solution contained DNA VII, XII and QII and templates CvII, CxII, CqII, V to inhX, X to inhQ and Q to inhV. However, the concentration of QII was ten times higher than that of VII or XII so that QII starts first and inhibits VII. Then, there will be less inhX because of the decreasing number of VII. Therefore, XII will make a lot of inhQ and the state will change from "QII" to "XII". As long as the reaction network works well, the cycle of states may continue to change like "QII" -> "XII" -> "VII" -> "QII"... The purpose of the experiment is to find out the best concentrations of CxII and CqII.

• Inhibit Direction: V-|Q-|X

The solution contained DNA VII, XII and QII and templates CvII, CxII, CqII, V to inhX, X to inhQ and Q to inhV. However, the concentration of QII was ten times higher than that of VII or XII so that QII starts first and inhibits XII. Then, there will be less inhV because of the decreasing number of XII. Therefore, VII will make a lot of inhQ and the state will change from "QII" to "VII". As long as the reaction networks work well, the cycle of states may continues to change like "QII" -> "XII" -> "VII" -> "QII"... The purpose of the experiment is to find out the best concentrations of CvII and CqII.

In this experiment, we found out the good conditions for V-|Q-|X. However, the results for V-|X-|Q was not accurate enough to use them in the trioscillator system. Therefore, we decided to do the V-|X-|Q experiment again.

### October 12th

• Inhibition Direction: V-|X-|Q

The purpose of the experiment is to find out the best conditions for the inhibition cycle V-|X-|Q. We made a ramp of concentrations of CxII and CqII.

As the result, we found out that 0.50μL of CxII was too small, but 1,00μL of that was too big. We decided to search for the best concentration of CxII between 0.50μL and 1.00μL.

### October 18th

• Inhibit Direction: V-|X-|Q

According to the experiment in October 12th, [CxII] = 25nM is too low and [CxII] = 50nM is too high. Therefore, we investigated the best concentration of CxII.

### October 19th

The purpose of this experiment is to find the best concentration of NBI, the nicking enzyme, because we got a new batch of NBI. We use the trioscillator system and observe which tube behaves the best.

As a result, lower concentration of NBI seemed to be good. That is because, if there are a lot of NBI, too much inhibitors are produced, and the reaction network does not oscillate.