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

本研究计划开发的生物传感器由三个器件组成。一种是探测器装置，它能感知特定的化学物质，由参与转录调节的蛋白质组成。第二种是存储装置，它以一种不可折叠的形式保存信息，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。将用于存储装置的可逆DNA特异性的DNA转化酶基因置于检测蛋白的控制之下。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，导致可逆DNA的转化。这一事件开启了报告基因的表达。以LacI和AraC为检测器件构建模型传感器，前者检测IPTG，后者检测阿拉伯糖。hin基因编码一种特定于H反转的DNA转化酶，位于LacI可抑制或AraC可诱导的启动子的下游。这些构建体被引入到大肠杆菌菌株EKK15中，其中鞭毛蛋白基因在H段启动子的控制下。然而，由于H段处于离位，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，产生了运动细胞，表明这些构建物可用于IPTG（或乳糖）和阿拉伯糖的传感器。剂量反应分析表明，这些传感器过于敏感，不能用于实际用途。为了找到更好的记忆装置，在大肠杆菌和沙门氏菌的基因组中寻找新的可逆dna。一些隐前噬菌体被发现含有有缺陷的可逆dna。

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.