Archives of Clinical Microbiology

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Glass Bead Transformation

The important factor considered while applying the glass bead method for fungal transformation was the size of the glass beads. The glass beads with a 0.45-0.52 mm diameter were found to be suitable for transformation. The ratio of a number of spores to the amount of glass beads is also crucial, and the ratio of 1:1 yielded better transformation efficiency. Prior to transformation, the plasmid DNA was linearized with EcoRI restriction enzyme. Polyethylene glycol (PEG 3500) was added to the transformation mixture at a final concentration of 5% wt/ vol and 0.1 M lithium chloride was also added to facilitate the DNA uptake. The use of PEG results in clumping of treated cells which facilitate the trapping of DNA, whereas the use of high concentration of lithium ions renders the cell walls permeable to DNA without the need for protoplasts, a method initially applied for transforming yeast, S. cerevisiae. A simple, inexpensive, and reproducible transformation method was developed for Gram-positive bacteria. It was based on agitation of bacterial protoplasts with glass beads in the presence of DNA and polyethylene glycol. By using this method, the introduction of pGK12 into protoplasts of several strains of Gram-positive bacteria was achieved.

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