Aoudi Amneh, Labiad Ossama, Igalouzene Ramdane, Mejri Ousséma, Sanchez Maxime, Sanchez Maxime
Tumor Escape Resistance and Immunity Department, Cancer Research Center of Lyon (CRCL), Lyon, France.
INSERM U1052, CNRS UMR 5286, Lyon, France.
Bio Protoc. 2024 Mar 5;14(5):e4945. doi: 10.21769/BioProtoc.4945.
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.
硅藻是多种具有特殊生物技术价值的化合物的来源。因此，将代谢流重定向到特定途径需要阐明基因的特定功能。硅藻中最常用的方法是基因枪转化法，这是一种非常昂贵且耗时的方法。使用以与天然染色体相当的拷贝数维持为闭环的附加体已成为一种有用的蛋白质表达遗传系统，可避免多拷贝插入、对表达的位置特异性影响以及非靶向基因的潜在敲除。这些附加体可以通过接合作用从细菌导入硅藻。在这里，我们描述了一种详细的基因表达方案，包括1）网关克隆策略和2）从细菌到硅藻的质粒转移的接合方案。
