Yeast recombinational cloning for heterologous biosynthesis of polyketides: a molecular microbiology laboratory module for undergraduate students

• Published in: Journal of microbiology & biology education 2023-12, Vol.24 (3)
• Main Authors: Bejenari, Mihaela, Nielsen, Line, Spedtsberg, Eva Mie Lang, Nielsen, Mikkel Rank, Pedersen, Tobias Bruun, Sørensen, Jens Laurids
• Format: Article
• Language: English
• Subjects: cloning; laboratory exercise module; plasmids; Saccharomyces cerevisiae; Tips and Tools; transformation; yeast
• ISSN: 1935-7877; 1935-7885
• DOI: 10.1128/jmbe.00242-22

Recombinant plasmids are essential tools in molecular biotechnology, and reliable plasmid assembly methods have, therefore, become a prerequisite for the successful cloning and transfer of genes. Among the multitude of available plasmid assembly strategies, homologous recombinational cloning in yeast has emerged as a cost-effective and relatively simple method. Since we use this method routinely in our group for assembling large plasmids with secondary metabolite gene clusters and for direct heterologous production of polyketides in , we developed an exercise module for undergraduate students where they would get hands-on experience with these molecular practices. The exercises target several molecular techniques, including PCR, restriction enzyme digestion, and yeast recombinational cloning. The students will learn about plasmid assembly and yeast transformation methods by performing these experiments while inherently acquiring new skills valuable for their subsequent laboratory work or projects.