肠道共生梭菌遗传操作工具箱

IF 4.4 2区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Pengjie Yang , Jinzhong Tian , Lu Zhang , Hui Zhang , Gaohua Yang , Yimeng Ren , Jingyuan Fang , Yang Gu , Weihong Jiang
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引用次数: 0

摘要

肠道微生物与人类健康密切相关,但仍有许多知识有待了解。共生梭菌是一种条件致病性人类肠道细菌,被认为是早期诊断肠道肿瘤的潜在生物标志物。然而,由于缺乏有效的工具箱,无法对这种细菌进行多样化的遗传操作,限制了对它的深入研究。在此,我们获得了共生菌的代表菌株共生菌 ATCC 14940 的完整基因组序列。在此基础上,我们进一步开发了该细菌的一系列遗传操作方法。首先,在鉴定出共生菌 ATCC 14940 中的功能性复制子 pBP1 之后,我们建立了一种高效的 DNA 共轭转移方法,从而能够将外源质粒快速引入细胞。接下来,我们构建了一个双质粒 CRISPR/Cas12a 系统,用于该细菌的基因组编辑,对大多数选定基因的抑制率超过 60%,并高效地删除了三个目标基因(90%)。最后,该工具箱被用于鉴定共生孢子菌 ATCC 14940 的关键功能基因,涉及其生长、重要代谢物的合成和毒力。我们的工作有效地建立并优化了肠道共生杆菌的基因组编辑方法,从而为该细菌的进一步基础研究和应用研究提供了有力支持。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A toolbox for genetic manipulation in intestinal Clostridium symbiosum

Gut microbes are closely related with human health, but remain much to learn. Clostridium symbiosum is a conditionally pathogenic human gut bacterium and regarded as a potential biomarker for early diagnosis of intestinal tumors. However, the absence of an efficient toolbox that allows diverse genetic manipulations of this bacterium limits its in-depth studies. Here, we obtained the complete genome sequence of C. symbiosum ATCC 14940, a representative strain of C. symbiosum. On this basis, we further developed a series of genetic manipulation methods for this bacterium. Firstly, following the identification of a functional replicon pBP1 in C. symbiosum ATCC 14940, a highly efficient conjugative DNA transfer method was established, enabling the rapid introduction of exogenous plasmids into cells. Next, we constructed a dual-plasmid CRISPR/Cas12a system for genome editing in this bacterium, reaching over 60 % repression for most of the chosen genes as well as efficient deletion (>90 %) of three target genes. Finally, this toolbox was used for the identification of crucial functional genes, involving growth, synthesis of important metabolites, and virulence of C. symbiosum ATCC 14940. Our work has effectively established and optimized genome editing methods in intestinal C. symbiosum, thereby providing strong support for further basic and application research in this bacterium.

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来源期刊
Synthetic and Systems Biotechnology
Synthetic and Systems Biotechnology BIOTECHNOLOGY & APPLIED MICROBIOLOGY-
CiteScore
6.90
自引率
12.50%
发文量
90
审稿时长
67 days
期刊介绍: Synthetic and Systems Biotechnology aims to promote the communication of original research in synthetic and systems biology, with strong emphasis on applications towards biotechnology. This journal is a quarterly peer-reviewed journal led by Editor-in-Chief Lixin Zhang. The journal publishes high-quality research; focusing on integrative approaches to enable the understanding and design of biological systems, and research to develop the application of systems and synthetic biology to natural systems. This journal will publish Articles, Short notes, Methods, Mini Reviews, Commentary and Conference reviews.
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