权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Development of biosensors using a memory device made from an invertible DNA

使用由可逆 DNA 制成的存储装置开发生物传感器

基本信息

批准号：
13554033
负责人：
KUTSUKAKE Kazuhiro
金额：
$ 8.9万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13554033/
关键词：
invertible DNA Memory device Biosensor Flagellar phase variation H segment Diphasic E. coli Reporter gene Cryptic prophage 転写スイッチアラビノースセンサー

项目摘要

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.

本研究计划开发的生物传感器由三个装置组成。一种是检测器装置，它可以检测特定的化学物质，由参与转录调节的蛋白质制成。第二种是记忆装置，它以不挥发的形式保存信息，由含有启动子的可逆DNA制成。第三种是报告基因，其表达由记忆装置中的启动子控制。将对用于存储装置的可转化DNA特异的DNA转化酶基因置于检测蛋白的控制下。当一种特定的化学物质与检测蛋白结合时，DNA转化酶的基因被诱导，导致转化体DNA的倒位。以LacI和AraC为检测器构建了模型传感器：前者可检测IPTG，后者可检测阿拉伯糖。hin基因，它编码的DNA转化酶特异性H反转，被放置在下游的启动子阻遏的LacI或诱导的AraC。将这些构建体导入大肠杆菌菌株EKK 15中，其中唯一的鞭毛蛋白基因在H区段中的启动子的控制下。然而，由于H片段处于关闭方向，鞭毛蛋白基因不表达，导致细胞的非运动表型。当将IPTG或阿拉伯糖添加到培养基中时，产生运动细胞，表明这些构建体可用于IPTG（或乳糖）和阿拉伯糖的传感器。为了寻找更好的记忆器件，我们在大肠杆菌和沙门氏菌的基因组中寻找新的可逆DNA。一些隐蔽的前噬菌体被发现含有缺陷的可逆DNA。