Allosteric Biosensor Screening Using Barcode Enrichment Sequencing
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Solution Overview
Problem
Existing allosteric transcription factor biosensors have a narrow range of molecules they can bind, limiting their applicability and functionality, and there is a need to develop methods to engineer these proteins for novel ligands.
Innovation Solution
A method of selecting allosteric biosensor proteins which bind a target ligand comprises providing a library of replicating plasmids comprising an expression construct and primer binding sites for next generation sequencing of at least a portion of an allosteric domain variant and a reporter, wherein the reporter is operably linked to a first promoter for expression of the allosteric protein variant or domain variant, and determining a fold enrichment for the allosteric protein or domain variant, and determining a subpopulation of variants with the highest fold enrichment as the selected allosteric biosensors, and selecting a specific measure of the efficacy of the allosteric domain variant or allosteric domain variant, and selecting a subpopulation of variants with the highest fold enrichment as the selected allosteric domain variant, and selecting a specific measure of the allosteric domain variant, and selecting a specific measure of the allosteric protein or domain variant.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If the ligand binding pocket of transcription factors is mutated to bind novel ligands, then the range of bound molecules is expanded, but the allosteric function is lost
Solution Approach 1:
The patent applies preliminary action by pre-evolving the ligand binding pocket through directed evolution to create variants that can bind novel ligands while preserving the allosteric communication pathway. The evolutionary process is conducted in stages, with selection for both ligand binding and allosteric function maintained throughout, preventing loss of the critical allosteric mechanism while expanding ligand specificity
Solution Approach 2:
The patent employs parameter changes by systematically varying amino acid residues in the ligand binding pocket while maintaining the overall protein structure and allosteric pathway integrity. This allows the binding pocket to adapt to new ligands through controlled changes in chemical properties (size, charge, hydrophobicity) without disrupting the conformational changes required for allosteric function
2Adaptability or versatility
If a library of allosteric protein variants is generated and screened, then new specificities are identified, but the complexity of the selection process increases
Solution Approach 1:
The patent applies universality by creating a standardized selection system where a single reporter gene construct can be used to screen for allosteric function across multiple different ligand types. The system universally detects allosteric activation through constitutive promoter-driven reporter expression, eliminating the need to design separate screening assays for each ligand and significantly reducing overall process complexity
Solution Approach 2:
The patent replaces complex mechanical screening methods with a biochemical reporter-based detection system. Instead of using labor-intensive physical assays to measure allosteric function, the system uses constitutive promoter activation and reporter gene expression as a readout, which can be easily quantified through standard molecular biology techniques, thereby simplifying the selection process
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method allows for the identification of allosteric biosensors with new specificities by generating a library of allosteric protein variants, mapping them to barcode sequences, and selecting variants with high fold enrichment, thereby expanding the range of molecules that can be bound by these proteins.
Implementation Method 1
aTF binding to the inducer causes an allosteric change that decreases affinity for the operator sequence, allowing downstream gene expression
Implementation Method 2
performing next generation sequencing to determine a quantity of each barcode in the target ligand total RNA
Data Source
AI summary
Described herein is a method of selecting allosteric biosensor proteins which bind a target ligand. The method includes providing a library of replicating plasmids each including an expression construct and a reporter, wherein each expression construct includes a gene encoding the allosteric protein variant and the reporter, wherein the reporter includes a barcode sequence for identification of the allosteric protein variant or allosteric domain variant. The method further includes mapping the variants in the library to the barcode sequence or sequences associated with the variant and assigning variant-barcode pairs, growing a population of cells transfected with the library of replicating plasmids in the presence of the target ligand and isolating target ligand total RNA and target ligand library plasmids; performing next generation sequencing to determine a quantity of each barcode in the target ligand total RNA, determining a fold enrichment for each allosteric protein variant or allosteric domain variant in the target ligand total RNA, and selecting a subpopulation of variants with the highest fold enrichment as the selected allosteric biosensors.