1997 Fiscal Year Final Research Report Summary
Real-time monitoring of the expression of two gen
| Project/Area Number |
07558103
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 試験 |
|---|
| Research Field |
Cell biology
|
|---|
| Research Institution | Nagoya University |
|---|
Principal Investigator |
ISHIURA Masahiro Nagoya University, Graduate School of Scie, Associate Professor, 大学院・理学研究科, 助教授 (20132730)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
ISHINO Yoshizumi Takara Shuzo Co., Principal investigator, 主任研究員
KONDO Takao Nagoya University, Graduate School of Scie, Professor, 大学院・理学研究科, 教授 (10124223)
|
|---|
| Project Period (FY) |
1995 – 1996
|
| Keywords | Bioluminescence / luciferase / gene expression / real-time monitoring / cyanobacteria / Synechococus |
|---|
| Research Abstract |
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.
|
