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.
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.
- 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.
- Transfect ~200 ng of DNA coding for a GPCR along with 10–250 ng of DNA coding for a ONE-GO biosensor.
- 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.
- 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.
- 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.
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.
The ONE-GO Biosensors Kit contains 10 plasmids. Please refer to the individual plasmid pages below for more details on each plasmid in this kit:
Accessory Plasmids
The following related plasmids are not included in the kit, but can be ordered separately:
- pcDNA3.1-mas-KB1691-Nluc
- pcDNA3.1-mas-KB2123-Nluc
- pcDNA3.1-cyto-KB1691-Nluc
- pLVX-SV40-Galpha-s ONE-GO
- pLVX-SV40-Galpha-i1 ONE-GO
- pLVX-SV40-Galpha-i3 ONE-GO
- pLVX-SV40-Galpha-oA ONE-GO
- pLVX-SV40-Galpha-q ONE-GO
- pLVX-SV40-Galpha-13 ONE-GO
- pLVX-hSyn-Galpha-i1 ONE-GO
- pLVX-hSyn-Galpha-i3 ONE-GO
- pLVX-hSyn-Galpha-oA ONE-GO
- pLVX-hSyn-Galpha-q ONE-GO
- pLVX-hSyn-Galpha-13 ONE-GO
- pLVX-hSyn-Galpha-s ONE-GO
- pLVX-CMV-Galpha-i3 G183S ONE-GO
- pLVX-CMV-IRESWT-Gbetagamma ONE-GO
- pLVX-CMV-Galpha-s-IRESWT-Gbetagamma ONE-GO
- pLVX-CMV-Galpha-i3-IRESWT-Gbetagamma ONE-GO
- pLVX-hSyn-Galpha-s-IRESWT-Gbetagamma ONE-GO
- pLVX-hSyn-Galpha-i3-IRESWT-Gbetagamma ONE-GO