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ONE-GO Biosensors Kit
(Kit # 1000000224 )

Depositing Lab:   Mikel Garcia-Marcos

This kit consists of ten ONE vector G protein Optical (ONE-GO) biosensor plasmids for the expression of BRET-based biosensors of G proteins activated by G-protein-coupled receptors (GPCRs).

This kit will be sent as individual bacterial stabs at room temperature.

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$430 USD + shipping
Available to academics and nonprofits only.
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Original Publication

Direct interrogation of context-dependent GPCR activity with a universal biosensor platform Janicot R, Maziarz M, Park J-C, Zhao J, Luebbers A, Green E, Philibert CE, Zhang H, Layne MD, Wu JC, Garcia-Marcos M. Cell. 2024 Feb 26; doi: 10.1016/j.cell.2024.01.028. PMID: 38412860. Article

Description

ONE vector G protein Optical (ONE-GO) biosensors are bioluminescence resonance energy transfer (BRET)-based biosensors of G proteins activated by GPCRs. This kit (and accessory plasmids) can be used to perform large-scale profiling of many GPCR modulatory conditions in parallel. Each plasmid expresses all the components of the biosensor in optimal proportions from a vector backbone that allows lentiviral packaging as a single payload. This one-vector design makes the kit suitable as a "turn-key" assay platform and supports implementation in poorly transfectable systems like primary cells.

Each of the plasmids in this kit encodes a biosensor for a different G protein, collectively targeting members of all four existing G protein families (Gs, Gi/o, Gq/11, and G12/13). Each construct consists of a Gα subunit tagged with YFP (at an internal location that does not perturb the G protein’s function) and a nanoluciferase detector module that specifically recognizes GTP-bound Gα. The Gα-YFP serves as a BRET acceptor when the detector module binds. Since GPCRs stimulate G proteins by promoting GTP loading on Gα, this biosensor system provides a direct readout of GPCR activity. Exogenous Gα-YFP assembles with endogenous Gβγ to form functional Gαβγ heterotrimers. This system provides robust responses with very low levels of expression of the exogenous components, ensuring fidelity and lack of interference.

The ONE-GO biosensor platform has been validated in transiently transfected cell lines with dozens of GPCRs, in stable cell lines with endogenous GPCRs, and upon acute transduction of cell lines or primary cells like neurons, cardiac fibroblasts, or endothelial cells, among others.

Two-panel illustration of the components and applications of ONE-GO biosensors. Left panel depicts a plasmid being introduced to a cell and the ONE-GO proteins that are expressed from it. GDP-Gα-YFP forms a complex with endogenous GPCR and Gβγ proteins. Arrows indicate that when the GDP is replaced with GTP, the nanoluciferase detector module binds GTP-Gα-YFP, placing Nluc close to YFP and generating BRET signal. A list indicates the G proteins targeted, which are also reflected by the plasmid names given in the Contents of Kit section. Right side depicts two applications, parallel GPCR phamacological profiling with a 96-well plate or context-dependent GPCR signaling in several tissue and cell types.
  • ONE-GO biosensors measure the formation of Gα-GTP by BRET. The sensors in this kit target ten different G proteins covering all four G protein families (Gs, Gi/o, Gq/11, G12/13). The biosensor consists of a YFP-tagged G protein and a nanoluciferase-fused detector protein that specifically binds to active (GTP-bound) Gα. These components are delivered as a single genetic payload by transfection or by lentiviral transduction. Once expressed in cells, the Gα-YFP assembles into functional heterotrimers with endogenous Gβγ, enabling detection of activation triggered by endogenous GPCR. Example applications include large-scale GPCR pharmacological profiling in cell lines or interrogation of context-dependent GPCR signaling in primary cells.

Kit Documentation

ONE-GO Biosensors Plasmid Sequences (DOCX, 55 KB)

Protocol

Using ONE-GO biosensors in transfectable cell lines:

The DNA amounts listed below are suggestions for Calcium-phosphate transfection in HEK293T cells. Amounts may need to be adjusted for other types of transfections/cell types.

For an initial experiment, we recommend doing a titration of ONE-GO biosensor DNA to determine what conditions work best in each individual lab.

  1. Transfect ~200 ng of DNA coding for a GPCR along with 10–250 ng of DNA coding for a ONE-GO biosensor.
  2. The following day, harvest cells and perform luminescence measurements by adding a nanoluciferase substrate to cells and simultaneously measuring YFP and Nluc emissions.

Using ONE-GO biosensors in primary cells:

The optimal conditions to transduce primary cells will need to be adjusted based on stock lentivirus concentration and cell type.

For an initial experiment, we recommend doing a titration of a lentivirus encoding a ONE-GO biosensor to find optimal transduction conditions.

  1. Transduce cells by diluting concentrated lentivirus encoding a ONE-GO sensor in cell culture media. We recommend dilutions in the range of 1:50–1:5000.
  2. Allow time for biosensor to be expressed in transduced cells (typically 24–48 h), then perform luminescence measurements by adding a nanoluciferase substrate to cells and simultaneously measuring YFP and Nluc emissions.

Additional Protocols:

Protocol for detecting endogenous GPCR activity in primary cell cultures using ONE-GO biosensors. Janicot R, Garcia-Marcos M. STAR Protoc. 2024 Oct 1; doi: 10.1016/j.xpro.2024.103355. PubMed Article

Protocol to investigate G protein-coupled receptor signaling kinetics and concentration-dependent responses using ONE-GO biosensors. Janicot R, Garcia-Marcos M. STAR Protoc. 2024 Oct 11; doi: 10.1016/j.xpro.2024.103383. PubMed Article

How to Cite this Kit

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.

For your Materials and Methods section:

“The ONE-GO Biosensors Kit was a gift from Mikel Garcia-Marcos (Addgene kit #1000000224).“

For your Reference section:

Direct interrogation of context-dependent GPCR activity with a universal biosensor platform Janicot R, Maziarz M, Park J-C, Zhao J, Luebbers A, Green E, Philibert CE, Zhang H, Layne MD, Wu JC, Garcia-Marcos M. Cell. 2024 Feb 26; doi: 10.1016/j.cell.2024.01.028. PMID: 38412860.

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