Optimization of a genetically encoded biosensor for cyclin B1-cyclin dependent kinase 1

Fluorescent protein (FP)-based biosensors have revolutionized the ability of researchers to monitor enzyme activities in live cells. While the basic design principles for FP-based biosensors are well established, first-generation biosensor constructs typically suffer from relatively low fluorescence...

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Bibliographic Details
Published in:Molecular bioSystems 2014-02, Vol.10 (2), p.191-195
Main Authors: Belal, Ahmed Saied F, Sell, Brittney R, Hoi, Hiofan, Davidson, Michael W, Campbell, Robert E
Format: Article
Language:English
Subjects:
Biosensing Techniques
CDC2 Protein Kinase - genetics
CDC2 Protein Kinase - metabolism
Cyclin B1 - genetics
Cyclin B1 - metabolism
Escherichia coli - genetics
Escherichia coli - metabolism
Fluorescence Resonance Energy Transfer
HeLa Cells
High-Throughput Screening Assays
Humans
Luminescent Proteins - chemistry
Luminescent Proteins - genetics
Luminescent Proteins - metabolism
Mitosis
Protein Engineering
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Summary:Fluorescent protein (FP)-based biosensors have revolutionized the ability of researchers to monitor enzyme activities in live cells. While the basic design principles for FP-based biosensors are well established, first-generation biosensor constructs typically suffer from relatively low fluorescence responses that limit their general applicability. The protein engineering efforts required to substantially improve the biosensor responses are often both labour and time intensive. Here we report the application of a high throughput bacterial colony screen for improving the response of kinase biosensors. This effort led to the development of a second-generation cyclin B1-CDK1 biosensor with a 4.5-fold greater response than the first-generation biosensor.
ISSN:1742-206X
1742-2051
DOI:10.1039/c3mb70402e