Real-time monitoring of the expression of two gen

实时监测二代表达

基本信息

批准号：
07558103
负责人：
ISHIURA Masahiro
金额：
$ 4.22万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07558103/
关键词：
Bioluminescence luciferase gene expression real-time monitoring cyanobacteria Synechococus Synechecocus 色突然変異

项目摘要

We attempted to monitor the expression of two genes simultaneously in viable cells.by using luciferases with different colors.Firstly, we tried to develop bacterial luciferases with different colors by mutagenesis in Escherichia coli. pUC7 plasmic carrying a luciferase luxAB gene set from Vibrio harvehyi was introduced into a mutagenic E.coli strain and the E.coli cells were cultured at various temperatures (25,30, or 37ﾟC) overnight to induce mutations in the luxAB genes. Plasmids were recovered from the mutagenic cells and reintroduced into a recAE.coli strain, HB101, and the HB101 cells were plated on LB plates containing ampicillin and cultured overnight at 37ﾟC to be allowed to develop colonies. The strength and color of bioluminescence of each colony were monitored by an automated colony-bioluminescence monitoring apparatus. We analyzed more than several ten thousands of colonies but could not obtain any clones with different color. Only non-bioluminescent clones or bioluminescent colonies with reduced light intensity were obtained. Thus, it might be difficult to isolate mutant bacterial luciferases with different colors.Then, we focused on firefly luciferases with different colors. we introduced the firefly luciferase luc gene into the cyanobacterium Synechococcus sp.strain PCC 7942 and confirmed the light emission of the transformed cells when luciferin was added to the cells exogenously. Next, luc genes with different colors were introduced into Synechococcus cells and confirmed the bioluminescence of the transformed cells. We developed a preliminary bioluminescence-monitoring machine to discrriminate different colors, but have not yet succeeded to discriminate different colors by this machine.

我们尝试通过使用不同颜色的荧光素酶在活细胞中同时监测两个基因的表达。首先，我们尝试在携带哈维弧菌荧光素酶luxAB基因集的大肠杆菌中通过诱变开发不同颜色的细菌荧光素酶。引入诱变大肠杆菌菌株，并将大肠杆菌细胞在不同温度（25，30，或37°C) 过夜以诱导 luxAB 基因突变从诱变细胞中回收质粒并重新引入recAE.coli 菌株 HB101，并将 HB101 细胞铺在含有氨苄青霉素的 LB 平板上并在 37°C 下培养过夜。我们分析了每个菌落的生物发光强度和颜色。超过数万个菌落，但无法获得任何具有不同颜色的克隆，仅获得非生物发光克隆或光强度降低的生物发光菌落，因此，分离具有不同颜色的突变细菌荧光素酶可能是困难的。重点研究了不同颜色的萤火虫荧光素酶，我们将萤火虫荧光素酶luc基因导入蓝藻聚球藻菌株PCC 7942中，并证实了其发光。接下来，将不同颜色的luc基因引入聚球藻细胞中，并证实了转化细胞的生物发光。我们开发了一种初步的生物发光监测机器来区分不同的颜色。通过这台机器成功区分不同的颜色。