TRUPATH
(Kit #
1000000163
)
Depositing Lab: Bryan Roth
A collection of 20 plasmids encoding alpha, beta, or gamma subunits of the heterotrimeric G protein complex for detection of heterotrimer dissociation.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome Olsen RHJ, DiBerto JF, English JG, Glaudin AM, Krumm BE, Slocum ST, Che T, Gavin AC, McCorvy JD, Roth BL, Strachan RT. Nat Chem Biol.2020 May 4. pii: 10.1038/s41589-020-0535-8. doi: 10.1038/s41589-020-0535-8. PubMed PMID: 32367019.
Description
The TRUPATH collection, deposited by Bryan Roth’s lab, consists of 20 plasmids encoding alpha (14 plasmids), beta (2 plasmids), or gamma (4 plasmids) subunits of the heterotrimeric G protein complex that can be used to detect heterotrimer dissociation by bioluminescence resonance energy transfer 2, or BRET2 (see illustration). Alpha subunits have Renilla luciferase (RLuc8) inserted at sites within their alpha-helical domain or third switch region, beta subunits are unmodified, and gamma subunits have GFP2 inserted at their N-termini. RLuc8 insertion sites and heterotrimer compositions were exhaustively engineered and evaluated to maximize BRET2 dynamic range for detecting G protein-coupled receptor agonist-induced heterotrimer dissociation.
TRUPATH measures heterotrimeric G protein dissociation by bioluminescence resonance energy transfer 2, or BRET2, between an alpha subunit-fused RLuc8 and gamma subunit-fused GFP2 that requires the substrate coelenterazine 400a or methoxy e-coelenterazine and is spatially- and orientationally- dependent. In the absence of drug and positive coupling to a G protein-coupled receptor, the alpha, beta, and gamma subunits of the heterotrimer are associated, producing a high BRET2 signal. In the presence of drug and with positive coupling, the alpha subunit and beta-gamma complex dissociate, producing a lower BRET2 signal. Over a range of drug concentrations, a concentration-response curve can be generated and fit to yield parameter estimates for drug efficacy and potency that are subject to minimal amplification and insensitive to receptor reserve. (This image was created using BioRender; the RLuc8 icon was generated in BioRender using Protein Data Bank entry 2PSD.)
Protocols
Agonist and antagonist TRUPATH assays for G protein-coupled receptors. DiBerto JF, Smart K, Olsen RHJ, Roth BL STAR Protoc.2022 Apr 1;3(2):101259. doi: 10.1016/j.xpro.2022.101259. PubMed PMID: 35403009.
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:
“TRUPATH was a gift from Bryan Roth (Addgene kit #1000000163).”
For your Reference section:
TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome Olsen RHJ, DiBerto JF, English JG, Glaudin AM, Krumm BE, Slocum ST, Che T, Gavin AC, McCorvy JD, Roth BL, Strachan RT. Nat Chem Biol.2020 May 4. pii: 10.1038/s41589-020-0535-8. doi: 10.1038/s41589-020-0535-8. PubMed PMID: 32367019.
TRUPATH - #1000000163
- Resistance Color Key
Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.
- Inventory
Searchable and sortable table of all plasmids in kit. The Well column lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.
- Kit Plate Map
96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.
Resistance Color Key
| Ampicillin |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | pcDNA5/FRT/TO-GAlphai1-RLuc8 | Ampicillin |
| A / 2 | pcDNA5/FRT/TO-GAlphai2-RLuc8 | Ampicillin |
| A / 3 | pcDNA5/FRT/TO-GAlphai3-RLuc8 | Ampicillin |
| A / 4 | pcDNA5/FRT/TO-GAlphaoA-RLuc8 | Ampicillin |
| A / 5 | pcDNA5/FRT/TO-GAlphaoB-RLuc8 | Ampicillin |
| A / 6 | pcDNA5/FRT/TO-GAlphaz-RLuc8 | Ampicillin |
| A / 7 | pcDNA5/FRT/TO-GAlphaGustducin-RLuc8 | Ampicillin |
| A / 8 | pcDNA5/FRT/TO-GAlphasS-RLuc8 | Ampicillin |
| A / 9 | pcDNA5/FRT/TO-GAlphasL-RLuc8 | Ampicillin |
| A / 10 | pcDNA5/FRT/TO-GAlphaQ-RLuc8 | Ampicillin |
| A / 11 | pcDNA5/FRT/TO-GAlpha11-RLuc8 | Ampicillin |
| A / 12 | pcDNA5/FRT/TO-GAlpha15-RLuc8 | Ampicillin |
| B / 1 | pcDNA5/FRT/TO-GAlpha12-RLuc8 | Ampicillin |
| B / 2 | pcDNA5/FRT/TO-GAlpha13-RLuc8 | Ampicillin |
| B / 3 | pcDNA3.1-Beta1 | Ampicillin |
| B / 4 | pcDNA3.1-Beta3 | Ampicillin |
| B / 5 | pcDNA3.1-GGamma1-GFP2 | Ampicillin |
| B / 6 | pcDNA3.1-GGamma8-GFP2 | Ampicillin |
| B / 7 | pcDNA3.1-GGamma9-GFP2 | Ampicillin |
| B / 8 | pcDNA3.1-GGamma13-GFP2 | Ampicillin |
Kit Plate Map - #1000000163
Accessory Plasmids
Ten additional accessory plasmids are also available. These plasmids are not included in the kit, but can be ordered separately: