Engineering fluorescent proteins for improved FRET performance and four-color biosensing
Abstract/Contents
- Abstract
- Engineered fluorescent proteins (FPs) have been widely used as biosensor components and protein tags. Improved and diversified variants continue to expand the FP toolbox, allowing us to re-design existing biosensors and develop new ones. In my thesis work, I present three new FPs and demonstrate the relevance of their improved properties in two applications. In the first example, I show that Clover and mRuby2, new green and red FPs engineered as a FRET (Förster Resonance Energy Transfer) pair, improve the performance of FRET-based reporters when substituted for traditional cyan FP (CFP) and yellow FP (YFP) pairs. Clover and mRuby2 improved photostability, FRET dynamic range, and emission ratio changes; and these improvements enhanced detection of transient biochemical events such as neuronal action potential firing and RhoA activation in growth cones. In the second example, I show that an engineered red FP, Maroon1, possessing a uniquely red-shifted excitation, enables the use of genetically encoded reporters in a fourth wavelength channel beyond the three common blue-cyan, green-yellow, and orange-red channels. In conjunction with the Fucci cell cycle reporter system, I used Maroon1 and the cyan FP, mTurquoise2, to label histone H1.0 and PCNA, respectively. Using this system, Fucci4, all four cell cycle stages can be visualized during live-cell imaging.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2015 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Lam, Amy |
|---|---|
| Associated with | Stanford University, Department of Bioengineering. |
| Primary advisor | Lin, Michael Z |
| Thesis advisor | Lin, Michael Z |
| Thesis advisor | Bryant, Zev David |
| Thesis advisor | Meyer, Tobias |
| Advisor | Bryant, Zev David |
| Advisor | Meyer, Tobias |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Amy Lam. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2015. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2015 by Amy Lam
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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