Real-time monitoring of gene expression by bioluminescence

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Analysis of gene expression is essential for all aspects of biology today. Real-time monitoring of gene expression from living cells is very powerful tool to dissect molecular mechanisms for many biological phenomena. To apply this technique to wide range of cells, a stable and versatile method need to be developed. In this project, we analyzed many technical problem that the in vivo real-time method should address.Bioluminescence from luciferase protein in the cell is affected by various factors, including subtrate, stability of enzyme, energy supply, etc. To avoid these factors, we applied two luciferase system of railroad worm to this purpose. This insect have two similar luciferase genes. While two luciferases share similar characteristics, waveform of bioluminescence is quite different each other (540 and 630 nm). With suppy of thses genes, we introduce them to cyanobacteria under different promoters. Then we developed automated bioluminescence detection system based on two photomultiplier tube and interference filters of different wave length. We finally succeeded to obtain cyanobacteria that contains two luciferase reporter and confirmed that siginals from two promoters can be detected without significant cross-talk. Therefore, now we can monitor expressions of two gene concurrently. With this method, we will enhance reliability to bioluminescence signals and evaluate gene expression in higher precision. Application of these reproter system can be expanded to various biological system to obtain reliable monitor of gene expression.