Plant Yeast Two-Hybrid Library Construction and Screening Service

We are committed to becoming your reliable assistant and partner in the field of plant protein

  1. Home
  2. Services
  3. Plant Molecular Biology Assay Services
  4. Plant Protein-Protein Interaction Analysis Services
  5. Plant Yeast Two-Hybrid Library Construction and Screening Service
Services
Online Inquiry

Plant Yeast Two-Hybrid Library Construction and Screening Service

Inquiry

CD BioSciences provides yeast two-hybrid (Y2H) library construction and screening services to help clients explore protein-mediated signaling pathways and regulatory networks in plants. Our extensive library construction experience and optimized total RNA extraction protocols ensure total RNA purity integrity and sample diversity.

The Y2H library is a large repository of all expressed genes, and these genes have been constructed onto prey vectors. In theory, the Y2H library can express all plant proteins, and screening experiments can be conducted by constructing the target genes onto bait vectors. Moreover, using the Y2H library to screen interaction proteins is more cost-effective than mass spectrometry.

Figure 1. Y2H screening steps. – CD BioSciences Figure 1. Y2H screening steps. (Mehla, J., et al., 2015)

Service Content

Y2H library construction service

We offer two technologies for Y2H library construction, Gateway recombination and SMART amplification. The cDNA libraries constructed by SMART are representative of the abundance of mRNA in the original samples, preserving the integrity of the biogenetic information. Gateway avoids the effects of PCR amplification, can be applied to cloning large fragments of genes, and allows the transfer of DNA between different expression vectors. 

Type Gateway SMART
RNA initiation > 300μg >> 1μg
Advantages High sample starting volume, low self-connection rate, high reorganization rate, and high code shift rate. Low sample starting volume, simple process, and low code shift rate.
  • Experimental content
  1. RNA extraction, including plant RNA extraction and mRNA isolation.
  2. cDNA library construction.

    3. a) cDNA synthesis and ligation.

    b) cDNA isolation by length.

    c) Recombination into yeast two-hybrid vector pGADT7 and electrotransformation.

    d) Library assay and plasmid extraction.

    e) Data collation and analysis.

Sample requirements Y2H library construction standard Y2H library delivery results
Cell count > 1×107 Raw library volume > 1×107CFU E. coli glycerol bacteria, 1mL/tube
Plant tissue > 2g The average insertion fragment > 1200bp Library plasmid > 400g
Plant seeds > 3g Positive cloning rate > 95% Library construction report and pictures

Y2H library screening service

Before yeast screening libraries, we provide clients with subcellular localization services on bait proteins, to clarify whether they are localized in the nucleus, cytoplasm, or membrane, to select corresponding libraries.

  • Experimental content
  1. Vector construction
  2. Validation of bait self-activation
  3. Library screening

    4. a) Co-transfer of bait and library plasmids.

    b) Optimization of yeast screening conditions.

    c) Sequencing of screening results.

    d) Rotary Validation

Sample requirements Y2H library screening delivery results
Bait gene sequences.
The plasmid containing bait genes, 2-5µg.
  1. Full sequence and annotation information of recombinant vector.
  2. Enzymatic mapping and sequencing results of the recombinant vector.
  3. Recombinant vector including one copy of plasmid and one copy of E. coli bacteriophage (25% glycerol bacteria).
  4. Experimental pictures and reports.
  5. Sequencing results of positive clones.
  6. Reverse validation of the prey plasmid.

CD BioSciences, a leading biological company in plant protein research, uses yeast two-hybrid systems to help clients explore protein-protein interactions in plants. If you need to research protein-mediated signaling pathways and regulatory networks in plants, please contact us and we will help you unravel the mechanisms of plant protein interactions and gain a deeper understanding of plant biology.

Reference

  1. Mehla, J., et al. (2015). The yeast two-hybrid system: a tool for mapping protein-protein interactions. Cold Spring Harbor protocols. (5), 425-430.

For research use only, not for clinical use.