Bacterial Two-Hybrid (B2H) Service for PPI Screening

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The Bacterial Two-Hybrid (B2H or BTH) system is a powerful, genetically-based method for detecting and screening these crucial interactions in vivo. As pioneers in the field, Creative Biolabs has refined the B2H system into a robust, high-throughput platform. Our services are designed to provide you with clear, reliable, and actionable data, accelerating your research from discovery to validation. We combine state-of-the-art technology with deep expertise to deliver customized solutions for even the most challenging PPI studies, including those involving membrane proteins and transcriptionally active proteins.

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The Principle of Our Advanced Bacterial Two-Hybrid (B2H) System

The B2H system is an ingenious method that leverages the modular nature of transcription factors within a prokaryotic host, typically E. coli. The core principle relies on the reconstitution of a functional transcription factor, which in turn activates the expression of a reporter gene, signaling a direct interaction between two proteins of interest.

Our primary system is based on the well-validated adenylate cyclase (CyaA) from Bordetella pertussis. Here's how it works:

Protein Fusion: The CyaA enzyme is split into two non-functional fragments, T25 and T18.
Bait and Prey Construction: Your "bait" protein (X) is genetically fused to one fragment (e.g., T25), creating the X-T25 fusion. The "prey" protein (Y) or a library of potential partners is fused to the other fragment, creating the Y-T18 fusion.
Interaction-Mediated Reconstitution: These fusion constructs are co-expressed in a cya-deficient E. coli strain. If the bait (X) and prey (Y) proteins interact, they bring the T25 and T18 fragments into close proximity.
Signal Activation: This proximity allows T25 and T18 to reconstitute a functional adenylate cyclase enzyme.
Reporter Gene Expression: The active enzyme synthesizes cyclic AMP (cAMP). The cAMP-catabolite activator protein (CAP) complex then binds to specific promoters, activating the transcription of reporter genes like lacZ (encoding β-galactosidase) or mal operons.
Phenotypic Detection: A positive interaction is easily identified by a distinct color change on indicator plates (e.g., blue colonies on X-Gal plates) or by the bacteria's ability to utilize specific sugars (e.g., maltose).

Workflow of a random Y2H library screening. (Creative Biolabs Original). Fig. 1 The bacterial adenylate cyclase two-hybrid assay (BACTH).¹

Key Advantages of Choosing the B2H System

While the Yeast Two-Hybrid (Y2H) system is well-known, the B2H system offers distinct and powerful advantages, making it the superior choice for many research applications.

Feature	Bacterial Two-Hybrid (B2H)	Yeast Two-Hybrid (Y2H)	Why It Matters for Your Research
Growth Rate & Speed	Very Fast (E. coli doubles ~20-30 min)	Slower (Yeast doubles ~90-120 min)	Faster library screening, quicker turnaround times, and reduced project timelines.
Transformation Efficiency	Extremely High	Moderate	Enables the screening of larger, more complex libraries, increasing the chance of discovering rare interactors.
Library Complexity	Superior representation of library clones	Prone to biases and loss of clones	Ensures a more comprehensive and unbiased screen.
Prokaryotic Proteins	Native Environment	Heterologous environment	Ideal for studying proteins from bacteria, archaea, or bacteriophages in their natural context.
Nuclear Localization	Not required; interactions occur in the cytoplasm	Proteins must be imported into the nucleus	Allows screening of proteins that are toxic or cannot be localized to the yeast nucleus, including many membrane proteins and transcription factors.
False Positives	Lower rate due to fewer endogenous transcription factors interfering with the assay.	Higher rate of false positives.	More reliable, higher-quality primary hits, reducing the downstream validation workload.
Membrane Proteins	Specialized systems available (e.g., BACTH)	Challenging	A key advantage for studying integral membrane proteins, receptors, and channels.

Our Comprehensive B2H Service Portfolio

At Creative Biolabs, we don't offer a one-size-fits-all solution. We provide a tailored suite of B2H services to meet your specific research goals, from one-on-one interaction analysis to large-scale library screening. Our Step-by-Step Service Workflow:

Step 1: Initial Consultation & Project Design

Our Ph.D.-level experts consult with you to understand your project goals, protein characteristics, and desired outcomes.
We help you select the optimal B2H system and strategy.

Step 2: Bait & Prey Vector Construction

Gene synthesis and codon optimization for expression in E. coli.
Subcloning of your target genes into our proprietary B2H bait and prey vectors.
Sequence verification of all constructs.

Step 3: Bait Characterization & Auto-activation Test

A critical quality control step. We express the bait protein alone to ensure it does not self-activate the reporter gene, which would lead to false positives.
We perform expression analysis via Western Blot to confirm bait protein stability.

Step 4: B2H Library Screening

We offer both custom library construction and screening of our premade, high-quality cDNA or ORF libraries (e.g., Human, Mouse, Virus).
High-efficiency co-transformation of the bait construct and the prey library into our specialized E. coli reporter strain.
Screening of up to 10^8 primary clones to ensure comprehensive coverage.

Step 5: Hit Identification and Validation

Positive colonies are selected based on phenotype (color or growth).
Prey plasmids from positive clones are isolated and the inserts are sequenced to identify the interacting partners.
Hits are categorized and ranked based on interaction strength and sequence data.

Step 6: Rigorous Hit Validation (Optional)

To eliminate false positives, we perform a re-array and one-on-one re-testing of putative interactions.
We can perform downstream validation using orthogonal methods like Co-Immunoprecipitation (Co-IP) or Surface Plasmon Resonance (SPR).

Step 7: Bioinformatics Analysis & Final Report

Identified interactors are analyzed using bioinformatics tools to classify them by function, pathway, and cellular location.
You receive a comprehensive report detailing the methodology, raw data, sequence of all interactors, bioinformatics analysis, and a conclusion from our experts.

Why Partner with Creative Biolabs?

Unmatched Expertise: With over 20 years in the industry, our scientific team possesses deep, practical knowledge of PPI analysis.
Cutting-Edge Technology: We continuously invest in and refine our B2H platforms to ensure the highest sensitivity and reliability.
Complete Customization: Your project is unique. We tailor every step of the workflow to your specific needs.
Speed and Efficiency: Our optimized protocols and high-throughput capabilities ensure rapid project completion.
Robust Quality Control: We implement multiple QC checkpoints, including bait auto-activation and expression tests, to guarantee data you can trust.
Transparent Communication: We provide regular updates and a dedicated project manager to guide you from start to finish.

Explore Our Related Services

The B2H system is a powerful discovery tool. We offer a comprehensive range of services to validate and further characterize your findings. Bacterial two-hybrid selection system from Creative Biolabs can not only study interactions of different proteins from both prokaryotes and eukaryotes but also apply successfully in the protein-DNA analysis, and identify antigen-specific single domain antibodies.

Other optional two-Hybrid systems:

Ready to uncover the critical protein interactions driving your research forward? Let the experts at Creative Biolabs accelerate your path to discovery.

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Reference

Hunke, Sabine, and Volker S. Müller. "Approaches to analyze protein–protein interactions of membrane proteins." Protein Interactions 10 (2012): 38069. Distributed under Open Access license CC BY 3.0, without modification. https://doi.org/10.5772/38069

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All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.