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

Single-copy genuttryck för karakterisering av multidrug effluxsystem

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.

Författare i fokus: Främja forskning om antibiotikaresistens med hjälp av en bakteriestam med brist på utflöde och ett genuttryckssystem med en kopia

Karakterisering av multidrug effluxsystem i Acinetobacter baumannii med hjälp av en bakteriestam med brist på efflux och ett genuttryckssystem med en kopia

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.

Framställning av elektrokompetenta celler för att uppnå genuttryck i en bakteriestam med brist på efflux 

Till att börja med placera alla sterila 1.5 milliliter mikrofugerör och sterila elektroporeringskyvetter på is. Ställ sedan flaskan med sterilt vatten, som förvarades vid fyra grader Celsius, på is. Överför 1,5 ml av den nattliga bakteriekulturen till ett av 1,5 milliliters mikrofugerören och centrifugera vid 13 000 G i två minuter för att pelletera cellerna.Använd en enmilliliters pipett för att ta bort all supernatant utan att störa cellpelleten. Tillsätt ytterligare 1,5 ml bakteriekultur i samma mikrofugerör, centrifugera vid 13 000 G i två minuter och ta bort supernatanten. Tillsätt en milliliter iskallt, sterilt vatten till cellpelleten och suspendera igen med försiktig pipettering tills pelleten inte längre sitter i botten av mikrofugeröret.Centrifugera de återsuspenderade cellerna vid 13 000 g i två minuter och avlägsna försiktigt supernatanten med en enmilliliterspipett. Blanda försiktigt om den sista cellpelleten i 200 mikroliter iskallt vatten och överför 100 mikroliter av den slutliga cellsuspensionen till det andra iskalla 1,5 milliliters mikrofugeröret.

Research

JoVE Journal

Immunology and Infection

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

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 ...

Electroporation Procedure for the Single&#45;Copy Gene Expression in an Efflux&#45;Deficient Bacterial Strain

Elektroporeringsprocedur för genuttryck i en bakteriestam med brist på utflöde