Kunoh Tatsuki, Ono Erika, Yamamoto Tatsuya, Suzuki Ichiro, Takeda Minoru, Nomura Nobuhiko
Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577 Japan.
Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577 Japan.
Bio Protoc. 2023 Apr 20;13(8):e4652. doi: 10.21769/BioProtoc.4652.
Genetic strategies such as gene disruption and fluorescent protein tagging largely contribute to understanding the molecular mechanisms of biological functions in bacteria. However, the methods for gene replacement remain underdeveloped for the filamentous bacteriaSP-6. Their cell chains are encased in sheath composed of entangled nanofibrils, which may prevent the conjugation for gene transfer. Here, we describe a protocol optimized for gene disruption through gene transfer mediated by conjugation withS17-1 with details on cell ratio, sheath removal, and loci validation. The obtained deletion mutants for specific genes can be used to clarify the biological functions of the proteins encoded by the target genes. Graphical overview.
基因破坏和荧光蛋白标记等遗传策略在很大程度上有助于理解细菌生物学功能的分子机制。然而，丝状细菌SP-6的基因替换方法仍未得到充分发展。它们的细胞链被包裹在由缠结的纳米纤维组成的鞘中，这可能会阻止基因转移的接合。在这里，我们描述了一种通过与S17-1接合介导的基因转移进行基因破坏的优化方案，详细介绍了细胞比例、鞘去除和位点验证。获得的特定基因缺失突变体可用于阐明靶基因编码的蛋白质的生物学功能。图形概述。
