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Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
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Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries.

Thomas C Scanlon1, Elizabeth C Gray, Karl E Griswold

  • 1Thayer School of Engineering, Dartmouth College, Hanover, USA. Thomas.C.Scanlon@Dartmouth.edu

BMC Biotechnology
|November 26, 2009
PubMed
Summary
This summary is machine-generated.

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Yeast homologous recombination libraries can be dominated by multiple vector transformants, with up to 90% of clones bearing multiple plasmids. These multiply transformed yeast cells persist, potentially skewing screening results and requiring careful identification.

Area of Science:

  • Molecular Biology
  • Yeast Genetics
  • Synthetic Biology

Background:

  • Recombinant microbial hosts are crucial for linking genotype to phenotype in library screening.
  • While multiple plasmid transformants are characterized in E. coli, quantitative data in yeast is lacking.
  • Saccharomyces cerevisiae is a key eukaryotic platform for cell surface display and protein engineering.

Purpose of the Study:

  • To characterize the prevalence and nature of multiple vector transformants in yeast libraries.
  • To assess the impact of these transformants on recombinant library screening.
  • To develop a method for identifying multiply transformed yeast cells.

Main Methods:

  • Analysis of plasmid-born gene libraries constructed by yeast homologous recombination.

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  • DNA sequencing to identify and quantify multiple vector transformants.
  • Assessment of transformant persistence during liquid outgrowth.

    • Main Results:

    • Up to 90% of clones in yeast homologous recombination libraries can be multiple vector transformants.
    • These clones often carry four or more unique mutant genes and persist for over 24 hours.
    • Multiple vector transformant frequency is influenced by vector concentration and ratio but not transformation efficiency.

    Conclusions:

    • Multiple vector transformants can dominate yeast libraries created via homologous recombination.
    • The prevalence and persistence of these cells have significant implications for yeast library screening.
    • Awareness and rapid sequencing methods are crucial for managing complications from multiply transformed yeast cells.