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Related Experiment Video

Updated: Jun 7, 2025

Competitive Genomic Screens of Barcoded Yeast Libraries
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Competitive Genomic Screens of Barcoded Yeast Libraries

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Workflow to Select Functional Promoter DNA Baits and Screen Arrayed Gene Libraries in Yeast.

Iris Fañanás-Pueyo1, Ana-Mariya Anhel1, Ángel Goñi-Moreno1,2

  • 1Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA/CSIC), Campus de Montegancedo UPM, Pozuelo de Alarcón (Madrid), Madrid, Spain.

Current Protocols
|November 21, 2024
PubMed
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This study presents a new workflow for identifying functional promoter fragments for yeast one-hybrid (Y1H) assays. The method uses phylogenetic analysis and high-throughput screening to improve the accuracy of DNA-protein interaction studies.

Area of Science:

  • Molecular Biology
  • Genetics
  • Bioinformatics

Background:

  • The yeast one-hybrid (Y1H) system is crucial for identifying DNA-protein interactions.
  • Using full gene promoters in Y1H assays can reduce accuracy and sensitivity, particularly in complex organisms.
  • Efficient identification of suitable promoter fragments is essential for reliable Y1H results.

Purpose of the Study:

  • To develop a robust workflow for identifying conserved, biologically relevant promoter fragments for Y1H assays.
  • To establish efficient high-throughput screening methods for DNA-protein interaction studies.
  • To facilitate the construction of gene regulatory networks.

Main Methods:

  • Phylogenetic analysis to identify conserved promoter fragments in Arabidopsis thaliana.
Keywords:
DNA–protein interactionarrayed librariesautomationone‐hybrid systemphylogenetic shadowing

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  • Manual and automated (robotized) high-throughput Y1H screening of arrayed open reading frame (ORF) libraries.
  • Application of the method for yeast two-hybrid (Y2H) screenings.

    • Main Results:

    • A workflow for selecting optimal DNA baits for Y1H screenings was established.
    • Efficient high-throughput screening protocols, including an automated version, were detailed.
    • The described methods are scalable and applicable to Y2H screenings for building gene regulatory networks.

    Conclusions:

    • The developed workflow enhances the identification of DNA-protein interactions using Y1H assays.
    • Phylogenetic analysis combined with high-throughput screening provides a powerful approach for selecting promoter fragments.
    • This methodology supports the comprehensive study of gene regulation and network construction.