On the application of 'seeding' techniques in the primary separation of plasmid DNA from neutralised E. coli lysates

Eirini Theodosiou*, Owen R T Thomas

*Corresponding author for this work

    Research output: Contribution to journalArticle

    Abstract

    Background: Initial extraction of plasmid DNA from Escherichia coli and its separation from host-derived contaminants is a difficult task to perform. Here, we examine the application of particle 'seeding' solid-liquid separation methods for primary recovery of plasmid DNA from neutralised alkaline cell lysates. Results: Planting magnetic particle 'seeds' during cell lysis resulted in enhanced phase separation, facile magnetic separation of the floc, slight improvements in plasmid purity, but diminished plasmid recoveries. When CaCO3-coated low-density microspheres were seeded into flocs, phase separation was impaired, shear-induced floc damage and contamination of the plasmid liquor with genomic DNA and cell debris occurred, but plasmid DNA recovery was improved. Introduction of hydrophobic low-density microspheres into the floc dramatically improved floc stiffness, phase separation and flotation efficiency, and reduced the solids content in the plasmid liquor 10-fold. However, strong reinforcement of the cell debris lattice by these microspheres hindered plasmid release into the liquor beneath. Conclusion: By incorporating magnetic or buoyant seeds during cell lysis we have identified new routes for separation of shear-sensitive cell debris solids from crude plasmid-containing liquors. Effective use of seeding approaches for difficult solid-liquid separation tasks will require evaluation of a wide range of seeds of varying architecture, size, shape, density and chemistry.

    Original languageEnglish
    Pages (from-to)192-200
    Number of pages9
    JournalJournal of Chemical Technology and Biotechnology
    Volume83
    Issue number2
    DOIs
    Publication statusPublished - 7 Jan 2008

    Fingerprint

    seeding
    plasmid
    Escherichia coli
    Plasmids
    DNA
    Microspheres
    Debris
    Phase separation
    Seed
    Recovery
    Magnetic separation
    Seeds
    Liquids
    Flotation
    lysis
    seed
    Reinforcement
    Contamination
    Stiffness
    Impurities

    Keywords

    • Alkaline lysis
    • Fillers
    • Flotation
    • Gene therapy
    • Genetic vaccination
    • Low-density gas-filled microspheres
    • Magnetic particle seeds

    Cite this

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    title = "On the application of 'seeding' techniques in the primary separation of plasmid DNA from neutralised E. coli lysates",
    abstract = "Background: Initial extraction of plasmid DNA from Escherichia coli and its separation from host-derived contaminants is a difficult task to perform. Here, we examine the application of particle 'seeding' solid-liquid separation methods for primary recovery of plasmid DNA from neutralised alkaline cell lysates. Results: Planting magnetic particle 'seeds' during cell lysis resulted in enhanced phase separation, facile magnetic separation of the floc, slight improvements in plasmid purity, but diminished plasmid recoveries. When CaCO3-coated low-density microspheres were seeded into flocs, phase separation was impaired, shear-induced floc damage and contamination of the plasmid liquor with genomic DNA and cell debris occurred, but plasmid DNA recovery was improved. Introduction of hydrophobic low-density microspheres into the floc dramatically improved floc stiffness, phase separation and flotation efficiency, and reduced the solids content in the plasmid liquor 10-fold. However, strong reinforcement of the cell debris lattice by these microspheres hindered plasmid release into the liquor beneath. Conclusion: By incorporating magnetic or buoyant seeds during cell lysis we have identified new routes for separation of shear-sensitive cell debris solids from crude plasmid-containing liquors. Effective use of seeding approaches for difficult solid-liquid separation tasks will require evaluation of a wide range of seeds of varying architecture, size, shape, density and chemistry.",
    keywords = "Alkaline lysis, Fillers, Flotation, Gene therapy, Genetic vaccination, Low-density gas-filled microspheres, Magnetic particle seeds",
    author = "Eirini Theodosiou and Thomas, {Owen R T}",
    year = "2008",
    month = "1",
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    doi = "10.1002/jctb.1834",
    language = "English",
    volume = "83",
    pages = "192--200",
    journal = "Journal of Chemical Technology and Biotechnology",
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    TY - JOUR

    T1 - On the application of 'seeding' techniques in the primary separation of plasmid DNA from neutralised E. coli lysates

    AU - Theodosiou, Eirini

    AU - Thomas, Owen R T

    PY - 2008/1/7

    Y1 - 2008/1/7

    N2 - Background: Initial extraction of plasmid DNA from Escherichia coli and its separation from host-derived contaminants is a difficult task to perform. Here, we examine the application of particle 'seeding' solid-liquid separation methods for primary recovery of plasmid DNA from neutralised alkaline cell lysates. Results: Planting magnetic particle 'seeds' during cell lysis resulted in enhanced phase separation, facile magnetic separation of the floc, slight improvements in plasmid purity, but diminished plasmid recoveries. When CaCO3-coated low-density microspheres were seeded into flocs, phase separation was impaired, shear-induced floc damage and contamination of the plasmid liquor with genomic DNA and cell debris occurred, but plasmid DNA recovery was improved. Introduction of hydrophobic low-density microspheres into the floc dramatically improved floc stiffness, phase separation and flotation efficiency, and reduced the solids content in the plasmid liquor 10-fold. However, strong reinforcement of the cell debris lattice by these microspheres hindered plasmid release into the liquor beneath. Conclusion: By incorporating magnetic or buoyant seeds during cell lysis we have identified new routes for separation of shear-sensitive cell debris solids from crude plasmid-containing liquors. Effective use of seeding approaches for difficult solid-liquid separation tasks will require evaluation of a wide range of seeds of varying architecture, size, shape, density and chemistry.

    AB - Background: Initial extraction of plasmid DNA from Escherichia coli and its separation from host-derived contaminants is a difficult task to perform. Here, we examine the application of particle 'seeding' solid-liquid separation methods for primary recovery of plasmid DNA from neutralised alkaline cell lysates. Results: Planting magnetic particle 'seeds' during cell lysis resulted in enhanced phase separation, facile magnetic separation of the floc, slight improvements in plasmid purity, but diminished plasmid recoveries. When CaCO3-coated low-density microspheres were seeded into flocs, phase separation was impaired, shear-induced floc damage and contamination of the plasmid liquor with genomic DNA and cell debris occurred, but plasmid DNA recovery was improved. Introduction of hydrophobic low-density microspheres into the floc dramatically improved floc stiffness, phase separation and flotation efficiency, and reduced the solids content in the plasmid liquor 10-fold. However, strong reinforcement of the cell debris lattice by these microspheres hindered plasmid release into the liquor beneath. Conclusion: By incorporating magnetic or buoyant seeds during cell lysis we have identified new routes for separation of shear-sensitive cell debris solids from crude plasmid-containing liquors. Effective use of seeding approaches for difficult solid-liquid separation tasks will require evaluation of a wide range of seeds of varying architecture, size, shape, density and chemistry.

    KW - Alkaline lysis

    KW - Fillers

    KW - Flotation

    KW - Gene therapy

    KW - Genetic vaccination

    KW - Low-density gas-filled microspheres

    KW - Magnetic particle seeds

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