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Overview
Zeocin™ is a member of the bleomycin/phleomycin family of antibiotics isolated from Streptomyces. It shows strong toxicity against bacteria, fungi (including yeast), plants and mammalian cell lines (Calmels et al., 1991; Drocourt et al., 1990; Gatignol et al., 1987; Mulsant et al., 1988; Perez et al., 1989). Since Zeocin™ is active in both bacteria and mammalian cell lines, vectors can be designed that carry only one drug resistance marker for selection.
Description
Zeocin™ is a formulation of phleomycin D1, a basic, watersoluble, copper-chelated glycopeptide isolated from Streptomyces verticillus. The presence of copper gives the solution its blue color. This copper-chelated form is inactive. When the antibiotic enters the cell, the copper cation is reduced from Cu2+ to Cu1+ and removed by sulfhydryl compounds in the cell. Upon removal of the copper, Zeocin™ is activated and will bind DNA and cleave it, causing cell death.
Resistance to Zeocin™
A Zeocin™ resistance protein has been isolated and characterized (Calmels et al., 1991; Drocourt et al., 1990). This 13,665 Da protein, the product of the Sh ble gene (Streptoalloteichus hindustanus bleomycin gene), binds stoichiometrically to Zeocin™ and inhibits its DNA strand cleavage activity. Expression of this protein in eukaryotic and prokaryotic hosts confers resistance to Zeocin™.
Handling Zeocin™
Concentrations of Zeocin™ to use for Selection
Zeocin™ and the Sh ble gene can be used for selection in mammalian cells (Drocourt et al., 1990; Mulsant et al., 1988), plants (Perez et al., 1989), yeast (Calmels et al., 1991; Gatignol et al., 1987), and prokaryotes (Drocourt et al., 1990). Suggested concentrations of Zeocin™ to use for selection in mammalian tissue culture cells, yeast, and E. coli are listed below.
Organism | Zeocin™ Concentration and Selective Medium |
---|---|
E. coli | 25-50 μg/ml in Low Salt LB medium* |
Yeast 50 | 50-300 μg/ml in YPD or minimal medium |
Mammalian cells | 50-1000 μg/ml (varies with cell line) |
*For efficient selection, the concentration of NaCl should not exceed 5 g/liter.
Introduction
Use 25-50 μg/ml of Zeocin™ for selection in E. coli. High salt and extremes in pH will inhibit the activity of Zeocin™ (see recommendations below).
Introduction
We have successfully transformed plasmids conferring Zeocin resistance into Saccharomyces cerevisiae and Pichia pastoris. The concentration of Zeocin™ required to select resistant transformants may range from 50 to 300 μg/ml, depending on the strain, pH, and ionic strength. Guidelines are provided below to assist you with selecting Zeocin™-resistant transformants.
We do not recommend spheroplasting for transformation of yeast with plasmids containing the Zeocin™ resistance marker. Spheroplasting involves removal of the cell wall to allow DNA to enter the cell. Cells must first regenerate the cell wall before they are able to express the Zeocin™ resistance gene. Plating spheroplasts directly onto selective medium containing Zeocin™ will result in complete cell death.
Transformation Method
We recommend electroporation, lithium cation protocols, or our EasyComp™ Kits for transformation of yeast with vectors that encode resistance to Zeocin™. Electroporation yields 103 to 104 transformants per μg of linearized DNA and does not destroy the cell wall of yeast. If you do not have access to an electroporation device, use chemical methods or one of the EasyComp™ Kits listed below.
Kit | Reactions | Catalog no. |
---|---|---|
S. c. EasyComp Transformation Kit (for Saccharomyces cerevisiae) | 6 x 20 transformations | K5050-01 |
Pichia EasyComp Transformation Kit (for Pichia pastoris) | 6 x 20 transformations | K1730-01 |
Ionic Strength and pH
Since yeast vary in their susceptibility to Zeocin™, we recommend that you perform a kill curve to determine the
lowest concentration of Zeocin™ needed to kill the untransformed host strain. In addition, the pH of the selection medium may affect the concentration of Zeocin™ needed to select resistant transformants. We recommend that you test media adjusted to different pH values (6.5 to 8) for the one that allows you to use the lowest possible concentration of Zeocin™.
Selection in Yeast
For successful selection of Zeocin™-resistant transformants we recommend the following: