Max Planck Partner Group, Faculty of Agriculture, Life and Environmental Sciences, Institute of Sericulture and Apiculture, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
Key Laboratory for Molecular Animal Nutrition, Ministry of Education, Hangzhou 310058, China.
Int J Mol Sci. 2022 May 13;23(10):5443. doi: 10.3390/ijms23105443.
As one of the most widespread groups of Gram-negative bacteria, bacteria are prevalent in almost all natural environments, where they have developed intimate associations with plants and animals. is a novel species of with clinical, animal, and plant-associated isolates, closely related to human and animal health, plant growth, and bioremediation. Although genetic manipulations have been proven as powerful tools for understanding bacterial biological and biochemical characteristics and the evolutionary origins, native isolates are often difficult to genetically manipulate, thereby making it a time-consuming and laborious endeavor. Here, by using the CRISPR-Cas system, a versatile gene-editing tool with a two-plasmid strategy was developed for a native strain isolated from the model organism silkworm () gut. We harmonized and detailed the experimental setup and clarified the optimal conditions for bacteria transformation, competent cell preparation, and higher editing efficiency. Furthermore, we provided some case studies, testing and validating this approach. An antibiotic-related gene, , was knocked out, resulting in the slow growth of the deletion mutant in LB containing chloramphenicol. Fusion constructs with knocked-in exhibited intense fluorescence. Altogether, the successful construction and application of new genetic editing approaches gave us more powerful tools to investigate the functionalities of the novel species.
作为革兰氏阴性菌中最广泛的群体之一，细菌普遍存在于几乎所有的自然环境中，它们与植物和动物形成了密切的联系。 是一种新型的 ，具有临床、动物和植物相关的分离株，与人类和动物健康、植物生长和生物修复密切相关。尽管遗传操作已被证明是了解细菌生物学和生物化学特性以及进化起源的有力工具，但天然分离株通常难以进行遗传操作，因此这是一项耗时费力的工作。在这里，我们使用 CRISPR-Cas 系统，通过两质粒策略为一种从模式生物家蚕肠道中分离出来的天然 菌株开发了一种多功能基因编辑工具。我们协调并详细说明了实验设置，并阐明了细菌转化、感受态细胞制备和更高编辑效率的最佳条件。此外，我们提供了一些案例研究，测试和验证了这种方法。敲除了一个与抗生素相关的基因 ，导致缺失突变体在含有氯霉素的 LB 中生长缓慢。带有敲入 的融合构建体表现出强烈的荧光。总之，新遗传编辑方法的成功构建和应用为我们提供了更强大的工具来研究新型 物种的功能。
