Transcription factors (TFs) are primary regulators of gene expression. Transcription is regulated by sequence-specific TFs that can bind to short genomic DNA elements located in promoters, enhancers, and other cis-regulatory modules. Yeast one-hybrid (Y1H) technology provides a gene-centered (DNA-to-protein) approach to identify the TFs capable of binding to the DNA fragment of interest. The Y1H assay is an extension of the yeast Two-Hybrid (Y2H) system. It is a powerful tool for studying nucleic acid-protein interactions and is essential for exploring gene regulatory mechanisms, identifying DNA-binding proteins, mapping promoter regions, and analyzing transcriptional networks.
With expertise and state-of-the-art technology, Amerigo Scientific provides high quality and reliable Y1H screening services to efficiently achieve the research objectives of customers. In Y1H assay, a DNA fragment is cloned upstream of two different reporter genes, and these reporter constructs are integrated into the genome of a yeast strain. Then, plasmids expressing hybrid proteins comprising a TF of interest fused to the strong transcriptional activation domain (AD) of yeast TF Gal4 are introduced into yeast strains. When a TF interacts with a DNA fragment of interest, the AD moiety activates reporter gene expression in yeast. Sequencing the plasmid in each of these colonies reveals the identity of the TFs that can bind the DNA fragment of interest.
Figure 1. Technical Principle of Our Yeast One-hybrid Screening
Key Advantages of Our Y1H Screening
- High-quality species-specific cDNA libraries cover more than 100 species.
- Up to 200bp of promoter sequences or pre-constructed bait plasmids for personalized solutions are provided.
- Comprehensive data includes BLAST annotations, GO and KEGG pathway analyses, and detailed experimental insights.
Content of Our Y1H Screening Service
Results are available in approximately two months, ensuring timely progress in client research.
| Content |
Timeline |
Core Deliverables |
Bait Construction
- Synthesize the target gene.
- Clone promoter sequences into the pHIS2 vector.
|
10 days |
- Positive Clone Sequences: Receive at least 15 high-quality sequences based on Sanger sequencing.
- Bait Plasmid (Optional): Return of the bait plasmid for further applications.
- NGS Sequencing Results: Comprehensive next-generation sequencing data with detailed bioinformatics analyses.
- Functional Annotation and Interaction Analysis: Includes GO and KEGG pathway enrichment and interaction strength evaluation.
- Detailed Experimental Report: Professionally prepared in Word format, featuring step-by-step methodologies, high-resolution images of results, and in-depth data interpretation.
|
Background Screening of Bait Plasmid
- Validate self-activation levels of the bait plasmid using 3AT background screening.
- Transform the bait plasmid into yeast strain Y187.
- Screen and optimize activation levels using selective SD-TLH medium.
|
7 days |
Yeast Transformation and Screening
- Co-transform bait and library plasmids into yeast.
- Optimize screening conditions using selective SD-TLH medium with 3AT.
- Sequence positive clones to identify DNA-binding candidates.
|
10 days |
Positive Clone Validation
- Conduct retesting and replicate experiments for confirmation.
- Perform data collation and analysis for high-confidence results.
|
5 days |
High-Throughput NGS and Analysis
- Perform next-generation sequencing on additional positive clones.
- Conduct GO and KEGG pathway enrichment analyses.
- Deliver in-depth annotated datasets with clear insights.
|
8 days |
Y1H One-to-One Validation Service
Inquiry
In addition to the Y1H screening service, Amerigo Scientific also provides a Y1H one-to-one validation service to meet the research needs for precise confirmation of specific DNA-protein interactions.
| Content |
Timeline |
Core Deliverables |
Gene synthesis with codon optimization and vector construction
- Construct protein A into pGADT7 vector.
- Construct the promoter into pHIS2 vector.
|
10 days |
- Recombinant plasmids (Optional)
- Detailed experimental report in Word format, including methodologies, step-by-step experimental details, high-resolution result images, and comprehensive data interpretation.
|
Transformation
- Transform recombinant plasmids into yeast cells.
- Perform background screening experiments.
|
7 days |
Interaction Validation
- Set up positive and negative control groups.
- Validate interactions between constructs.
|
10 days |
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