Mobilization of Plasmids from Bacteria into Diatoms by Conjugation Technique

BERDUN, FEDERICO; VALIÑAS, MATÍAS; PAGNUSSAT, GABRIELA; EDUARDO JULIAN ZABALETA

Bio-protocol

Bio-protocol LLC

Año: 2024 vol. 14

Diatoms serve as a source for a variety of compounds with particular biotechnological interest. Therefore, redirecting the flow to a specific pathway requires the elucidation of the gene’s specific function. The most commonly used method in diatoms is biolistic transformation, which is a very expensive and time-consuming method. The use of episomes that are maintained as closed circles at a copy number equivalent to native chromosomes has become a useful genetic system for protein expression that avoids multiple insertions, position-specific effects on expression, and potential knockout of non-targeted genes. These episomes can be introduced from bacteria into diatoms via conjugation. Here, we describe a detailed protocol for gene expression that includes 1) the gateway cloning strategy and 2) the conjugation protocol for the mobilization of plasmids from bacteria to diatoms.