preparation of electrocompetent cells to achieve single&#45;copy gene expression in an efflux&#45;deficient bacterial strain

用于表征多药物外排系统的单拷贝基因表达

Acinetobacter baumannii is a World Health Organization top priority pathogen due to its encompassing resistance to all classes of antibiotics1. It is an opportunistic pathogen mostly affecting hospitalized, injured, or immunocompromised people. A. baumannii largely evades antibiotics via efflux pumps, the most relevant being the Resistance-Nodulation-Division (RND) family of exporters2. Understanding how these efflux pumps work mechanistically will allow one to develop targeted therapeutic options.
One common way that cellular processes can be specifically distinguished is through genetic manipulation. However, the tools available for A. baumannii genetic studies are limited, and to further confound experimental design, clinical isolates often are resistant to the antibiotics routinely used for selection in genetic manipulations3. A second hurdle encountered when studying efflux pumps specifically is that they are strictly regulated-often by unknown factors-making it difficult to accurately isolate and attribute function to a single pump4. Seeing this need to expand the research toolbox, we developed a mini-Tn7-based, single-copy-insertion, inducible expression system that incorporates a Flp recombinase target (FRT) cassette, which allows for the removal of the selection marker5,6,7 (Figure 1). First created for Pseudomonas8,9,10, this elegant cloning and expression system was used to generate single-copy efflux pump complements into an RND efflux pump-deficient strain of A. baumannii (ATCC 17978::&#916;adeIJK,&#916;adeFGH,&#916;adeAB: hereafter referred to as A. baumannii AB258) that we generated11. Being able to study one efflux pump at a time and not overwhelm the bacterial cells with high-copy expression (as generally seen with plasmid-based expression systems), one can better learn about the critical, physiological aspects of each efflux pump with minimal interference and reduced complications.
This article describes how to use the mini-Tn7 system to complement a deleted gene of interest, RND efflux pump adeIJK, into the chromosome of A. baumannii AB258 through a series of uncomplicated steps performed over the course of 9 days7. The first set of steps re-introduces the deleted efflux pump genes cloned into the mini-Tn7-based insertion plasmid (Figure 2A) at the single attTn7 insertion site downstream of the well-conserved glmS gene (Figure 3A). This process is facilitated by a non-replicative helper plasmid (Figure 2B) that encodes for the transposase genes needed for Tn7-driven insertion. The second set of steps uses an excision plasmid (Figure 2C) for Flp recombinase-mediated removal of the gentamicin gene flanked by FRT sites (Figure 3B) to create an unmarked strain. Though this system is used to elucidate the essential roles and possible inhibitors of RND efflux pumps with respect to antibiotic resistance, it can be used to investigate any gene of interest.

作者聚焦：使用外排缺陷型细菌菌株和单拷贝基因表达系统推进抗生素耐药性研究

使用外排缺陷细菌菌株和单拷贝基因表达系统表征 鲍曼不动杆 菌的多药外排系统

We study multi-drug resistance efflux systems in gram-negative bacteria. We are interested in learning about the natural functions of these pumps and how these pumps can be used as a potential drug target. Expression of multi-drug resistance efflux systems is tightly regulated, making it difficult to study their functions.Thus, an expression system that bypasses the regulatory system can be quite useful in the characterization of these pumps. This protocol allows for the expression of genes at levels usually seen in natural settings. Thus, the activity of the gene products mimics what is observed in nature.

制备电感受态细胞以在外排缺陷细菌菌株中实现单拷贝基因表达 

首先，将所有无菌的 1.5 毫升微量离心管和无菌电穿孔比色皿放在冰上。然后将一瓶储存在四摄氏度的无菌水放在冰上。将 1.5 毫升过夜细菌培养物转移到 1.5 毫升微量离心管之一中，并以 13， 000 G 离心两分钟以沉淀细胞。使用一毫升移液管，在不干扰细胞沉淀的情况下除去所有上清液。将另外1.5毫升细菌培养物加入同一微量离心管中，以13,000G离心两分钟并除去上清液。向细胞沉淀中加入一毫升冰冷的无菌水，并用轻柔的移液重新悬浮，直到沉淀不再位于微量离心管的底部。将重新悬浮的细胞以13,000G离心两分钟，并使用一毫升移液管小心地除去上清液。轻轻地将最终细胞沉淀重新悬浮在 200 微升冰冷无菌水中，并将 100 微升最终细胞悬浮液转移到第二个冰冷的 1.5 毫升微量离心管中。

preparation-electrocompetent-cells-to-achieve-single-copy-gene

Research

JoVE Journal

Immunology and Infection

Preparation of Electrocompetent Cells to Achieve Single&#45;Copy Gene Expression in an Efflux&#45;Deficient Bacterial Strain

62 次观看.  University of Manitoba. 首先，将所有无菌的 1.5 毫升微量离心管和无菌电穿孔比色皿放在冰上。然后将一瓶储存在四摄氏度的无菌水放在冰上。将 1.5 毫升过夜细菌培养物转移到 1.5 毫升微量离心管之一中，并以 13， 000 G 离心两分钟以沉淀细胞。使用一毫升移液管，在不干扰细胞沉淀的情况下除去所有上清液。将另外1.5毫升细菌培养物加入同一微量离心管中，以13,000G离心两分钟并除去上清液?...

视频: 制备电感受态细胞以在外排缺陷细菌菌株中实现单拷贝基因表达 

Characterizing Multidrug Efflux Systems in Acinetobacter baumannii Using an Efflux&#45;Deficient Bacterial Strain and a Single&#45;Copy Gene Expression System

Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend the mechanisms behind this resistance to design new and effective therapeutic options. Unfortunately, our ability to investigate these mechanisms in A. baumannii is hindered by the paucity of suitable genetic manipulation tools. Here, we describe methods for utilizing a chromosomal mini-Tn7-based system to achieve single-copy gene expression in an A. baumannii strain that lacks functional RND-type efflux mechanisms. Single-copy insertion and inducible efflux pump expression are quite advantageous, as the presence of RND efflux operons on high-copy number plasmids is often poorly tolerated by bacterial cells. Moreover, incorporating recombinant mini-Tn7 expression vectors into the chromosome of a surrogate A. baumannii host with increased efflux sensitivity helps circumvent interference from other efflux pumps. This system is valuable not only for investigating uncharacterized bacterial efflux pumps but also for assessing the effectiveness of potential inhibitors targeting these pumps.

We describe a facile procedure for the single-copy chromosomal complementation of an efflux pump gene using a mini-Tn7-based expression system into an engineered efflux-deficient strain of Acinetobacter baumannii. This precise genetic tool allows for controlled gene expression, which is key for the characterization of efflux pumps in multidrug resistant pathogens.

Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend ...