通过革兰氏阴性细菌的负向选择评估质粒稳定性

Damián  Lobato Márquez; Laura  Molina García

doi:10.21769/BioProtoc.2261

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Peer-reviewed

Evaluation of Plasmid Stability by Negative Selection in Gram-negative Bacteria

通过革兰氏阴性细菌的负向选择评估质粒稳定性

Damián Lobato Márquez email

Laura Molina García

发布: 2017年05月05日第7卷第9期 DOI: 10.21769/BioProtoc.2261 浏览次数: 11987

评审: Valentine V TrotterAlba BlesaSeda Ekici

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Abstract

Plasmid stability can be measured using antibiotic-resistance plasmid derivatives by positive selection. However, highly stable plasmids are below the sensitivity range of these assays. To solve this problem we describe a novel, highly sensitive method to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells. The assay proposed here is based on an aph-parE cassette. When synthesized in the cell, the ParE toxin induces cell death. ParE synthesis is controlled by a rhamnose-inducible promoter. When bacteria carrying the aph-parE module are grown in media containing rhamnose as the only carbon source, ParE is synthesized and plasmid-containing cells are eliminated. Kanamycin resistance (aph) is further used to confirm the absence of the plasmid in rhamnose grown bacteria.

Keywords: Plasmid stability (质粒稳定性)

ParE (ParE)

Toxin (毒素)

Negative selection (负向选择)

Gram-negative bacteria (革兰氏阴性菌)

Background

lassically, plasmid stability has been measured by positive selection using antibiotic-resistance plasmid derivatives. Cells harbouring the studied plasmid are positively selected in the presence of the selection antibiotic (Gerdes et al., 1985; del Solar et al., 1987). The main drawback of this technique is its sensitivity; highly stable plasmids are below the sensitivity of these assays. To solve this problem alternative methods relying on the direct selection of plasmid-free cells such as the tetAR-chlortetracycline system, have been described (Bochner et al., 1980; Maloy and Nunn, 1981; Garcia-Quintanilla et al., 2006). Limitations of the tetAR-chlortetracycline method include poor reproducibility and the frequent occurrence of false positives (Li et al., 2013). Here, we describe a novel, highly sensitive plasmid stability assay based on the counter-selection of plasmid-containing cells. This assay is based on a cassette containing a ParE toxin-encoding gene controlled by a rhamnose-inducible promoter and a kanamycin resistance gene (aph) (Figure 1) (Maisonneuve et al., 2011). ParE is the toxin of the toxin-antitoxin system parDE, and targets the DNA gyrase, blocks DNA replication and induces DNA breaks leading to cell death (Jiang et al., 2002). The aph-parE cassette is inserted into the plasmid of interest using homologous recombination. Upon induction of PparE in minimal media containing rhamnose as the only carbon source, only plasmid-free cells survive (Lobato-Marquez et al., 2016). Kanamycin is then used to confirm the loss of the plasmid (Figure 2).

Figure 1. Scheme showing the integration process of the aph-parE cassette into the plasmid of interest. (1) The aph-parE cassette is first amplified by PCR using pKD267 plasmid as template. (2) Then, cells harbouring a plasmid encoding λ-Red recombinase are electroporated with aph-parE DNA fragment. λ-Red recombinase directs the specific integration of the aph-parE module into the plasmid region containing the 50 bp upstream and 50 bp downstream homologous sequences included in the oligos used for PCR. (3) Confirm the aph-parE insertion by using primers annealing with the cassette (red arrows) and with the plasmid (black arrows).

Figure 2. Plasmid stability procedure. To avoid plasmid loss, the strain carrying the aph-parE cassette is initially grown under antibiotic selection pressure (using kanamycin). When kanamycin is removed from the medium, the plasmid of interest will be lost after a certain number of generations. Plasmid-free cells are selected when the culture is plated in M9-rhamnose plates containing rhamnose as the only carbon source. Modified from Lobato-Marquez et al., 2016.

Materials and Reagents

Pipette tips
1.5 ml Eppendorf tubes
Millipore 0.22 µm pore size filter (EMD Millipore, catalog number: SLGP033RS )
9-cm sterile Petri dishes
Electroporation cuvettes 0.2 cm gap (Bio-Rad Laboratories, catalog number: 1652082 )
Glass beads for bacterial plating (VWR, catalog number: 201-0279 )
pKD267 plasmid (described in Maisonneuve et al., 2011)
pKD46 plasmid (described in Datsenko and Wanner, 2000)
Expand High Fidelity DNA polymerase (Roche Molecular Systems, catalog number: 11732641001 )
DpnI restriction enzyme (New England Biolabs, catalog number: R0176 )
PCR purification kit (5Prime, catalog number: 2300610 )
Plasmid purification kit (Roche Molecular Systems, catalog number: 11754777001 )
4 °C chilled sterile distilled MilliQ water
Distilled MilliQ water
Ampicillin sodium salt (Sigma-Aldrich, catalog number: A0166 )
L-arabinose (Sigma-Aldrich, catalog number: A3256 )
Kanamycin (Sigma-Aldrich, catalog number: K1876 )
D-glucose (Sigma-Aldrich, catalog number: G8270 )
Bacto tryptone (BD, Bacto^TM, catalog number: 211705 )
Bacto yeast extract (BD, Bacto^TM, catalog number: 288620 )
Sodium chloride (NaCl) (VWR, BDH^®, catalog number: 102415K )
European bacteriological agar (Conda, catalog number: 1800 )
Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: 71645 )
Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P9791 )
Ammonium chloride (NH₄Cl) (Sigma-Aldrich, catalog number: A9434 )
Calcium chloride dihydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: C3881 )
Magnesium sulfate (MgSO₄) (VWR, BDH^®, catalog number: 291175X )
Vitamin B1 (Thiamine) (Sigma-Aldrich, catalog number: T4625 )
L-rhamnose monohydrate (Sigma-Aldrich, catalog number: R3875 )
Potassium chloride (KCl)
Na₂HPO₄·12H₂O
Glycerol (v/v) (Sigma-Aldrich, catalog number: G5516 )
Luria-Bertani (LB) broth medium (see Recipes)
Luria Bertani plates (see Recipes)
M9 (10x) (see Recipes)
CaCl₂/MgSO₄ solution (100x)
M9 minimum medium (see Recipes)
M9-rhamnose plates (see Recipes)
Phosphate saline buffered (PBS) (see Recipes)
10% sterile glycerol (see Recipes)
L-rhamnose (see Recipes)

Equipment

Selection of single channel pipettes (2 µl, 20 µl, 20 µl, 1,000 µl) (Gilson, P-2, P-20, P-200, P-1000)
Glass 100 ml flasks (Fisher Scientific, Fisherbrand)
Glass 50 ml flasks (Fisher Scientific, Fisherbrand)
MiniSpin^® Eppendorf benchtop centrifuge (Eppendorf, model: MiniSpin^® )
Bacterial shaking incubator (Eppendorf, New Brunswick, model: Innova^® 4000 )
MicroPulser^TM electroporator (Bio-Rad Laboratories, model: MicroPulser Electroporator , catalog number: 1652100)
Eppendorf refrigerated centrifuge (Eppendorf, model: 5804 )
Spectrophotometer (Thermo Fisher Scientific, Thermo Scientific^TM, model: SPECTRONIC^TM 200 )
Milli-Q^® integral water purification system for ultrapure (Nanopure) water
DNA SpeedVac (Savant Systems, model: SpeedVac DNA 110 )
Autoclave (Prestige Medical, catalog number: 210004 )

Software

GraphPad Prism Software (https://www.graphpad.com/scientific-software/prism/)

Procedure

English

中文翻译

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如何引用

Lobato-Márquez, D. and Molina-García, L. (2017). Evaluation of Plasmid Stability by Negative Selection in Gram-negative Bacteria. Bio-protocol 7(9): e2261. DOI: 10.21769/BioProtoc.2261.