[Frontiers in Bioscience 17, 1931-1939, January 1, 2012]

Application of cell-free expression of GFP for evaluation of microsystems

Takahiko Nojima1, Shohei Kaneda2, Hiroshi Kimura2, Takatoki Yamamoto3, Teruo Fujii2

1College of Liberal Arts and Sciences, Kitasato University, Kanagawa 252-0373, Japan, 2Institute of Industrial Science, University of Tokyo, Tokyo 153-8505, Japan, 3Department of Mechanical and Cntrol Engineering, Tokyo Institute of Technology, Tokyo 152-8550, Japan


1. Abstract
2. Introduction
3. Application of cell-free GFP-expression as a report gate in biomolecular logic gate system
4. Application of cell-free GFP-expression as a reporter in on-chip capillary electrophoresis fractionation of DNA
5. Summary and perspective
6. Acknowledgements
7. References


Coupled cell-free transcription-translation (CFTT) of green fluorescent protein (GFP) has been applied as a reporter system to microfluidic chip-related technologies. In polymerase chain reaction (PCR)-based biomolecular logic gate system, in which addition of primer set and amplification of PCR product represent input and output signal respectively, GFP gene was inserted in the template DNA, which was then amplified, transcribed and translated to GFP. The green fluorescence reported as if the amplification has occurred or not, that is, the fluorescence reports positive output signal. CFTT of GFP was also adopted to evaluate on-chip capillary electrophoresis (CE)-based DNA fractionation, which was developed to isolate single DNA species from reaction mixture of DNA ligase-catalyzed DNA-assembly. As a model system, GFP gene was inserted in the target DNA fragment. The collected fraction was amplified with PCR and subjected to a CFTT system, and green fluorescence was observed showing that the fractionation was successful. These results showed that CFTT of GFP is a useful tool to verify, estimate, and monitor microfluidic chip-related technologies in which cell-free protein synthesis is involved.