Bis-(3′-5′)-cyclic dimeric guanosine monophosphate (cyclic di-GMP or c-di-GMP) is one of the most widespread and most intensely studied bacterial second messengers. C-di-GMP is found in bacteria and in mammalian cells, which plays prominent roles in bacterial physiological processes and mammalian innate immune responses. In bacteria, c-di-GMP regulates biofilm formation, motility, cell cycle, differentiation, virulence, and other processes. Most c-di-GMP-dependent signaling pathways control the ability of bacteria to interact with other cells or abiotic surfaces. C-di-GMP plays a key role in many bacterial lifestyle changes, including the transition from a motor state to a sessile state, which contributes to the establishment of multicellular biofilm communities. In addition, c-di-GMP regulates virulence of animal and plant pathogens, and virulence related processes controlled by c-di-GMP include host cell adhesion, secretion of virulence factors, cytotoxicity, invasion, antioxidant stress, and host immune response regulation. In mammals, c-di-GMP produced endogenously in response to foreign DNA or invading bacterial pathogens is involved in an innate immunity pathway, which is responsible for the surveillance of cytoplasmic DNA. Modulating c-di-GMP signaling pathways in bacteria may be a novel way to control biofilm formation and dispersal in medical and industrial settings. C-di-GMP that can be recognized by the mammalian immune system is considered a promising vaccine adjuvant.
Spinach™ Aptamer-based Cyclic Di-GMP Assay
Amerigo Scientific offers the spinach™ aptamer-based c-di-GMP assay to measure c-di-GMP levels in cells. The assay is a homogenous, simple mix-and- read, highly selective, HTS-ready assay that can be used to detect c-di-GMP produced in any biochemical or enzymatic reaction and to monitor intracellular c-di-GMP concentrations in cell-based applications.
For detection of c-di-GMP levels in cells or in any biochemical or enzymatic reaction
100 rxn
A sample is incubated with the reagents in the assay kit for 30 minutes, after which the fluorescence can be read on a fluorescent plate reader with a FITC/GFP filter or excitation wavelength of 482nm and emission wavelength of 505nm.
Features
Easy to use, save time
Quick response and good signal stability
Broad dynamic range
High selectivity (50nM of c-di-GMP)
Compatibility with cell-based assays without pelleting, lysis, or wash steps
Assay Principle
Spinach is a novel genetically encodable probe that fluorescently labels RNA in living cells. Spinach is a short RNA tag that can be inserted into the target RNA sequence and activates the fluorescence of otherwise non-fluorescent dyes, such as DFHBI and its derivative DFHBI-1T. DFHBI is a mimic of green fluorescent protein (GFP) fluorophore. Upon bound to Spinach, the highly fluorescent state of DFHBI is turned on. DFHBI and DFHBI-1T are cell permeable and have negligible toxicity in living cells. Compared to DFHBI, DFHBI-1T has lower background fluorescence and exhibits stronger overall brightness in living cells.
Spinach can be an indispensable tool for imaging intracellular RNA activity. Tagging an RNA with Spinach for fluorescent imaging does not require protein translation. Spinach can be used to visualize mRNAs in living cells and can also be used to image non-coding RNAs. Because of the inherent flexibility of RNA, Spinach can be designed as sensors to detect specific targets of interest. The evolution of the fluorescent signal is dependent on the initial binding of c-di-GMP to the c-di-GMP riboswitch in the sensor. The stable Spinach aptamer in turn binds to DFHBI-1T to produce fluorescence.
Kit Contents
Reagent
Amount
Storage
c-di-GMP Sensor
400 µg
-80°C
c-di-GMP
6.9 g
-20°C
DFHBI-1T Fluorophore
160.2 g
-20°C
4X c-di-GMP Assay (CA) Buffer
10 mL
25°C
4X Bacterial Compatibility (BC) Reagent
10 mL
25°C
RNase-Free Water
15 mL
25°C
C-di-GMP Estimation in Bacteria
E. coli cells expressing wild-type WspR (diguanylate cyclase from Pseudomonas aeruginosa) were treated with the antibiotic nitrofurazone and the vector dimethylformamide (DMF), or left untreated (control). The bacteria at different cell densities were added to plates to simulate varying c-di-GMP levels. Without any pelleting, washing, lysis, or organic extraction, c-di-GMP concentrations in bacterial cells grown in 96-well plates were measured by Spinach™ Aptamer-based Cyclic Di-GMP Assay. The results showed that the c-di-GMP concentration in bacteria varied with antibiotic treatment and bacterial density and showed that the presence of antibiotics or carriers did not affect the performance of the Aptamer-based Cyclic Di-GMP Assay.
C-di-GMP Standard Curve
C-di-GMP Estimate in WT WspR Cells
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