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优化CRISPR干扰系统用于研究Pseudomonas alloputida与球微生物组组合有关的基因

Marissa N Roghair Stroud^1,2, Dua X Vang^1,2, Larry J Halverson^1,2

¹Department of Plant Pathology, Entomology, and Microbiology, Iowa State University, Ames, Iowa 50011, United States.

ACS synthetic biology

|August 20, 2024

概括

我们为Pseudomonas alloputida KT2440开发了一种CRISPR干扰 (CRISPRi) 系统,使合成生物学和根球研究中的精确基因沉默成为可能. 这种工具有效调节基因表达和微生物相互作用在不同的环境.

科学领域:

合成生物学合成生物学
微生物生态学微生物生态学
细菌遗传学细菌遗传学

背景情况:

Pseudomonas alloputida KT2440 是合成生物学和研究根植殖的关键生物体.
在这种底盘中精确调节基因的现有工具是有限的,阻碍了先进的研究.

研究的目的:

开发和描述一种新型的CRISPR干扰 (CRISPRi) 系统,用于Pseudomonas alloputida KT2440.
为了实现精确的基因表达调制,用于研究微生物相互作用和工业应用.

主要方法:

一个基于迷你Tn7的转子子系统被设计为提供一个编码子优化的dCas9和sgRNA表达向量.
三种不同的促进体系统 (XylS/Pm,LacI/Plac,AraC/PBAD) 被整合并对基因抑制效率进行比较.
该系统的功能在树叶球殖民和调节pyoverdine生产中进行了测试.

主要成果:

开发的CRISPRi系统证明了在P.alloputida.中有效和可调节的基因沉默.
抑制必需基因导致植物根上的细菌细胞显著减少 (10-100倍).
通过CRISPRi介导的pvdH抑制消除了pyoverdine的产生,改变了P.alloputida抑制其他微生物的能力.

结论:

新的CRISPRi系统提供了一个强大的工具,用于在实验室和环境环境中对P. alloputida进行基因操纵.
该系统有助于对基因功能,微生物相互作用的研究,以及对微生物工程应用的开发.
模块化设计允许适应各种实验条件和未来的高通量研究,如CRISPRi-seq.

关键词:

这就是CRISPR干扰的原因.伪白属全白属 KT244040 的病毒.在XylS/Pm中使用.微生物 - 微生物相互作用.皮奥维尔迪因 (pyoverdine) 是一种的物质.根系球 (Rhizosphere) 是一个根系球.

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