Toolkit/mTOR pathway specific rapamycin biosensor

mTOR pathway specific rapamycin biosensor

Multi-Component Switch·Research

Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.

Summary

Using this new BRET system, we also designed a mTOR pathway specific rapamycin biosensor by integrating the FRB and FKBP12 protein dimerization system.

Usefulness & Problems

No literature-backed usefulness or problem-fit explainer has been materialized for this record yet.

Published Workflows

Novel BRET combination for detection of rapamycin-induced protein dimerization using luciferase from fungus Neonothopanusnambi.

2024

Objective: Develop a new orthogonal fungal luciferase-based BRET system and apply it to a rapamycin-inducible protein dimerization biosensor for mammalian cell culture.

Why it works: The workflow pairs an orthogonal fungal luciferase donor with red fluorescent protein acceptors to identify an efficient BRET combination, then couples that readout to rapamycin-induced FRB/FKBP12 dimerization so ligand-triggered association can be detected by BRET.

bioluminescence resonance energy transferrapamycin-induced FRB/FKBP12 protein dimerizationfusion-pair constructionacceptor comparisonbiosensor design in mammalian cell culture

Stages

  1. 1.
    BRET donor-acceptor pair construction and comparison(broad_screen)

    This stage identifies which acceptor red fluorescent protein performs best with the fungal luciferase donor before building a downstream biosensor.

    Selection: BRET efficiency of fused fungal luciferase and red fluorescent protein pairs

  2. 2.
    Rapamycin biosensor design using FRB/FKBP12(functional_characterization)

    This stage applies the selected BRET combination to a biologically targeted inducible dimerization sensor.

    Selection: ability of the new BRET system to report rapamycin-induced FRB/FKBP12 dimerization in an mTOR pathway-specific biosensor format

  3. 3.
    Mammalian cell culture demonstration of specificity and efficacy(confirmatory_validation)

    This stage confirms that the engineered BRET combination works in the intended mammalian cell culture application context.

    Selection: specificity and efficacy of the fungal luciferase-based BRET combination in mammalian cell culture

Steps

  1. 1.
    Create fused fungal luciferase and red fluorescent protein pairsBRET donor/acceptor fusion constructs

    Generate candidate BRET pairs using the fungal luciferase donor and red fluorescent protein acceptors.

    Fusion pairs had to be created before their BRET efficiency could be compared and before a biosensor could be built on the best-performing pair.

  2. 2.
    Compare BRET efficiency across acceptor pairs and identify tdTomato as best performerBRET pair candidates under comparison

    Select the highest-efficiency acceptor pairing for downstream biosensor construction.

    The best-performing acceptor had to be identified before applying the new BRET system to rapamycin biosensor design.

  3. 3.
    Integrate FRB and FKBP12 dimerization modules into the new BRET system to build a rapamycin biosensorengineered biosensor construct

    Convert rapamycin-induced FRB/FKBP12 dimerization into a BRET-detectable mTOR pathway-specific biosensor signal.

    The biosensor was built using the new BRET system after establishing a workable donor/acceptor combination.

  4. 4.
    Demonstrate specificity and efficacy of the fungal luciferase-based BRET combination in mammalian cell cultureBRET system and biosensor under validation

    Confirm that the engineered BRET combination functions effectively and specifically in the intended mammalian cell culture context.

    Cell-culture validation follows system construction and biosensor design to confirm practical applicability.

Taxonomy & Function

Primary hierarchy

Mechanism Branch

Architecture: A composed arrangement of multiple parts that instantiates one or more mechanisms.

Target processes

No target processes tagged yet.

Validation

Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication

Observations

successMammalian Cell Lineapplication demo

Inferred from claim c4 during normalization. The new fungal luciferase-based BRET combination showed specificity and efficacy for application in mammalian cell culture. Derived from claim c4. Quoted text: We demonstrated the specificity and efficacy of the new fungal luciferase-based BRET combination for application in mammalian cell culture

Source:

Supporting Sources

Ranked Claims

Claim 1application performancesupports2024Source 1needs review

The new fungal luciferase-based BRET combination showed specificity and efficacy for application in mammalian cell culture.

We demonstrated the specificity and efficacy of the new fungal luciferase-based BRET combination for application in mammalian cell culture
Claim 2comparative performancesupports2024Source 1needs review

Among the tested acceptor red fluorescent proteins, tdTomato provided the highest BRET efficiency with the fungal luciferase donor.

of which tdTomato provided the highest BRET efficiency
Claim 3design claimsupports2024Source 1needs review

The authors designed an mTOR pathway specific rapamycin biosensor by integrating the FRB and FKBP12 protein dimerization system into the new BRET system.

Using this new BRET system, we also designed a mTOR pathway specific rapamycin biosensor by integrating the FRB and FKBP12 protein dimerization system.
Claim 4future use potentialsupports2024Source 1needs review

The fungal luciferase-based BRET combination may enable multiplexed BRET assessment in the future.

that will provide the unique opportunity to perform multiplexed BRET assessment in the future
Claim 5tool capabilitysupports2024Source 1needs review

Neonothopanus nambi fungal luciferase can function as a new BRET donor when fused with acceptor red fluorescent proteins.

We demonstrated the possibility of using the fungal luciferase as a new BRET donor by creating fused pairs with acceptor red fluorescent proteins

Approval Evidence

1 source2 linked approval claimsfirst-pass slug mtor-pathway-specific-rapamycin-biosensor
Using this new BRET system, we also designed a mTOR pathway specific rapamycin biosensor by integrating the FRB and FKBP12 protein dimerization system.

Source:

application performancesupports

The new fungal luciferase-based BRET combination showed specificity and efficacy for application in mammalian cell culture.

We demonstrated the specificity and efficacy of the new fungal luciferase-based BRET combination for application in mammalian cell culture

Source:

design claimsupports

The authors designed an mTOR pathway specific rapamycin biosensor by integrating the FRB and FKBP12 protein dimerization system into the new BRET system.

Using this new BRET system, we also designed a mTOR pathway specific rapamycin biosensor by integrating the FRB and FKBP12 protein dimerization system.

Source:

Comparisons

No literature-backed comparison notes have been materialized for this record yet.

Ranked Citations

  1. 1.
    StructuralSource 1MED2024Claim 1Claim 2Claim 3

    Extracted from this source document.