Single-step Marker Switching in Schizosaccharomyces pombe Using a Lithium Acetate Transformation Protocol

Area of Science:

• Molecular Biology
• Yeast Genetics

Background:

• Traditional selectable markers in Schizosaccharomyces pombe, such as ura4+ and kanMX6, limit multi-gene studies.
• Introducing new markers often necessitates de novo strain construction, which is time-consuming.

Purpose of the Study:

• To develop an efficient, single-step protocol for marker switching in Schizosaccharomyces pombe.
• To enable the exchange of existing selectable markers for a wider range of options, simplifying genetic manipulation.

Main Methods:

• Utilized transformation cassettes with flanking homologies to existing markers (ura4+ and kanMX6).
• Employed lithium acetate transformation for introducing new markers into established fission yeast strains.
• Demonstrated the exchange of ura4+ for kanMX6, natMX4, or hphMX4, and MX markers for nutritional markers (arg3+, his3+, leu1+, ura4+).

Main Results:

• Successfully implemented a single-step marker switching protocol in Schizosaccharomyces pombe.
• Enabled the efficient replacement of ura4+ with antibiotic resistance markers (kanMX6, natMX4, hphMX4).
• Facilitated the substitution of MX markers with various nutritional markers, expanding genetic tool availability.

Conclusions:

• The developed protocol significantly simplifies and accelerates the process of marker manipulation in Schizosaccharomyces pombe.
• This method provides a versatile approach for researchers to adapt existing strains for diverse genetic studies, including multi-gene tagging and mutation.
• The availability of efficient marker switching enhances the utility of Schizosaccharomyces pombe as a model organism for genetic research.