Chapter title |
Measuring Cyclic Diguanylate (c-di-GMP)-Specific Phosphodiesterase Activity Using the MANT-c-di-GMP Assay.
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Chapter number | 20 |
Book title |
c-di-GMP Signaling
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Published in |
Methods in molecular biology, January 2017
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DOI | 10.1007/978-1-4939-7240-1_20 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7239-5, 978-1-4939-7240-1
|
Authors |
Eli, Dorit, Randall, Trevor E, Almblad, Henrik, Harrison, Joe J, Banin, Ehud, Dorit Eli, Trevor E. Randall, Henrik Almblad, Joe J. Harrison, Ehud Banin |
Abstract |
The second messenger, cyclic diguanylate (c-di-GMP), regulates a variety of bacterial cellular and social behaviors. A key determinant of c-di-GMP levels in cells is its degradation by c-di-GMP-specific phosphodiesterases (PDEs). Here, we describe an assay to determine c-di-GMP degradation rates in vitro using 2'-O-(N'-methylanthraniloyl)-cyclic diguanylate (MANT-c-di-GMP). Additionally, a protocol for the production and purification of recombinant Pseudomonas aeruginosa RocR, a c-di-GMP-specific PDE that may serve as a control in MANT-c-di-GMP assays, is provided. The use of the fluorescent MANT-c-di-GMP analogue can deliver fundamental information about PDE function, and is suitable for identifying and investigating c-di-GMP-specific PDE activators and inhibitors. |
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