• Abstract

We all have a biological clock which controls the periodicity of many physiological functions such as blood pressure, body temperature and concentration of hormones. The systems that generate circadian rhythms are called oscillator. Oscillator has been researched for deepening our knowledge of circadian rhythms, understanding genetic network and signal transfer.

Oscillator is expected to be used for various applications. It needs to be available in various types and can be made and controlled easily.

However, there are several problems of existing oscillator using protein.

1. There are only a few types of repressor proteins, which make it harder to make a sophisticated circuit in one cell.

2. Protein requires several steps for maturation, which makes it difficult to design and control oscillator.

The first problem of protein oscillator is the small type of the protein. Because the number of repressor proteins and promoters are limited, the variation of protein oscillator that can be made in one cell is not many.

Another problem of protein oscillator is the component, the protein. Protein requires several steps; transcription and translation, protein modification, folding, and degradation. When we want to construct or control such protein oscillator, we have to genetically engineer to change the transcriptional efficiency, translational efficiency and degradation ability. Therefore, it is difficult to design such gene circuits that can express the repressors periodically.

For the solutions of these problems and for the applications, we decided to construct the New type of oscillator circuit – RNA Oscillator.　We named the E. coli which has this function TwinklE. Coli.

　Whereas there are only a few types of proteins that can be used for construction of protein oscillator, Many kinds of RNA oscillators can be constructed by changing a few base pair. Therefore, it will allow us to construct a multiple and orthogonal oscillators in one cell.

Another advantage is that, although protein has several steps until degradation, RNA does not have the steps of translation, modification and degradation that are needed for proteins, and RNAs degradation occurs fast. Therefore, RNA oscillator can be designed and controlled by only changing the transcriptional efficiency and binding ability. It is easy to change the binding ability of RNA because all we have to do is to alter the base pair, and hence change the strength of base pairs binding.

The feature of our RNA oscillator is using Hammerhead ribozyme (HHR). HHR is a ribozyme which has the ability of self-cleavage. When self-cleavage occurs, the downstream sequence from the cleavage site is cleaved into smaller pieces. We constructed RNA oscillator by making HHR whose self-cleavage activity can be controlled by RNA binding.

Additionally, we expect the RNA oscillator to be put into practice by combination with diverse mechanisms. For example, we can make systems of automatic control with counter system, change output from RNA to protein with reporter circuit, and reset the oscillation with light sensor. We think that the combination with RNA oscillator can be applied for drug delivery and bioremediation.