HARAYAMA Shigeaki Marine Biotechnology Institute, Kamaishi Laboratory, Director, 釜石研究所, 所長
OHNISHI Kouhei Kochi University, Research Institute of Molecular Genetics, Associate Professor, 遺伝子実験施設, 助教授 (50211800)
TOMINAGA Akira OKAYAMA UNIVERSITY, Faculty of Science, Research Associate, 理学部, 助手 (60155570)
|Budget Amount *help
¥13,900,000 (Direct Cost: ¥13,900,000)
Fiscal Year 2003: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2002: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 2001: ¥8,900,000 (Direct Cost: ¥8,900,000)
The biosensor, which was planned to be developed in this study, consists of three devices. One is a detector device, which senses a specific chemical, and is made from a protein involved in transcription regulation. Second is a memory device, which saves information in an involatile form, and is made from an invertible DNA containing a promoter. Third is a reporter gene, whose expression is controlled by the promoter in the memory device. A gene for DNA invertase specific for the invertible DNA used for the memory device is placed under the control of the detector protein. When a specific chemical binds to the detector protein, the gene for DNA invertase is induced, resulting in inversion of the invertibe DNA. This event turns on the expression of the reporter gene.
Model sensors were constructed using LacI and AraC as detector devices : the former can detect IPTG, while the latter arabinose. The hin gene, which encodes a DNA invertase specific for H inversion, was placed downstream of the promoter repressible by LacI or inducible by AraC. These constructs were introduced into an Escherichia coli strain EKK15, in which a sole flagellin gene is under the control of the promoter in the H segment. However, because the H segment is in the off orientation, the flagellin gene, is not expressed, resulting in the nonmotile phenotype of the cell. When IPTG or arabinose was added to the medium, motile cells were produced, indicating that these constructs can be used for sensors of IPTG (or lactose) and arabinose. Dose-response analysis revealed that these sensors are too sensitive to use for the practical purpose.
In order to find better memory devices, novel invertible DNAs were searched in the genomes of Escherichia coli and Salmonella. Some cryptic prophages were found to contain defective invertible DNAs.