Improvements of protoplast transformation in Staphylococcus carnosus

Protoplast transformation is so far the only method to transfer plasmids into Staphylococcus carnosus. However, the preparation of new protoplasts for each transformation is time-consuming and the quality of the protoplasts may also vary each time. An improved method is described which allows the storage of the protoplasts deep frozen (at − 70°C) for several months without affecting their transforming capability. The possibility of keeping protoplasts competent for a long period opens several advantages: the transformation can be carried out immediately, the transformation procedure is reduced to only a couple of minutes, and the protoplasts of one batch have the same quality, yielding a more constant transformation frequency.