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

Çoklu İlaç Akış Sistemlerini Karakterize Etmek için Tek Kopyalı Gen İfadesi

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.

Yazar Spotlight: Akış Eksikliği Olan Bir Bakteri Suşu ve Tek Kopyalı Gen Ekspresyon Sistemi Kullanarak Antibiyotik Direnci Araştırmasını İlerletmek

Acinetobacter baumannii'de Akış Eksikliği Olan Bir Bakteri Suşu ve Tek Kopyalı Gen Ekspresyon Sistemi Kullanılarak Çoklu İlaç Akış Sistemlerinin Karakterize Edilmesi

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.

Akış eksikliği olan bir bakteri suşunda tek kopya gen ekspresyonu elde etmek için elektroyetkin hücrelerin hazırlanması 

Başlamak için tüm steril, 1,5 mililitrelik mikrofüj tüplerini ve steril elektroporasyon küvetlerini buzun üzerine yerleştirin. Daha sonra dört santigrat derecede saklanan steril su şişesini buzun üzerine koyun. Gece boyunca bakteri kültürünün 1.5 mililitresini 1.5 mililitrelik mikrofüj tüplerinden birine aktarın ve hücreleri peletlemek için iki dakika boyunca 13.000 G'de santrifüjleyin.Bir mililitrelik bir pipet kullanarak, hücre peletini bozmadan tüm süpernatanı çıkarın. Aynı mikrofüj tüpüne 1.5 mililitre bakteri kültürü daha ekleyin, iki dakika boyunca 13.000 G'de santrifüjleyin ve süpernatanı çıkarın. Hücre peletine bir mililitre buz gibi, steril su ekleyin ve pelet artık mikrofüj tüpünün dibinde kalmayana kadar hafif pipetleme ile yeniden süspanse edin.Yeniden süspanse edilmiş hücreleri iki dakika boyunca 13.000 G'de santrifüjleyin ve bir mililitrelik bir pipet kullanarak süpernatanı dikkatlice çıkarın. Son hücre peletini 200 mikrolitre buz gibi, steril suda nazikçe yeniden süspanse edin ve 100 mikrolitre son hücre süspansiyonunu ikinci buz gibi soğuk 1.5 mililitrelik mikrofüj tüpüne aktarın.

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 Görüntüleme.  University of Manitoba. Başlamak için tüm steril, 1,5 mililitrelik mikrofüj tüplerini ve steril elektroporasyon küvetlerini buzun üzerine yerleştirin. Daha sonra dört santigrat derecede saklanan steril su şişesini buzun üzerine koyun. Gece boyunca bakteri kültürünün 1.5 mililitresini 1.5 mililitrelik mikrofüj tüplerinden birine aktarın ve hücreleri peletlemek için iki dakika boyunca 13.000 G'de santrifüjleyin.Bir mililitrelik bir pipet kullanarak, hücre peletini bozmadan tüm süpernata...

Video: Akış eksikliği olan bir bakteri suşunda tek kopya gen ekspresyonu elde etmek için elektroyetkin hücrelerin hazırlanması 

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 ...