Minimal shuttle vectors for Saccharomyces cerevisiae.

Area of Science:

• Synthetic Biology
• Molecular Biology
• Yeast Genetics

Background:

• Advanced genetic engineering in *Saccharomyces cerevisiae* is limited by the availability of suitable vector backbones.
• Existing plasmids often contain unnecessary sequences, increasing size and reducing ligation efficiency.
• Restriction sites outside the multiple cloning site (MCS) are frequently unavailable for use.

Purpose of the Study:

• To design and validate a collection of minimal shuttle vectors for *Saccharomyces cerevisiae*.
• To provide smaller, more efficient plasmids for complex genetic engineering tasks.
• To increase the number of available restriction sites for molecular cloning.

Main Methods:

• De novo gene synthesis was used to construct six minimal integrating or centromeric shuttle vectors.
• Vectors were designed to include only essential elements: yeast and bacterial selection markers, origin of replication, and an MCS.
• Truncated variants of elements and elimination of non-essential sequences were employed to minimize plasmid size.

Main Results:

• A series of six minimal shuttle vectors (pLS series) were created, ranging from ~2.6 to 3.5 kb, the smallest reported for yeast.
• Each vector features ten unique restriction sites within the MCS and approximately 30 additional "free" cut sites.
• The new vectors are significantly smaller (63% on average) than commonly used pRS vectors, improving ligation efficiency.

Conclusions:

• The pLS vector series offers a valuable resource for advanced synthetic biology and genetic engineering in *S. cerevisiae*.
• These minimal vectors simplify complex procedures like protein domain grafting and multi-gene fusions.
• The reduced size and increased availability of restriction sites enhance cloning efficiency and experimental design.