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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   

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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

  1. Pipette tips  
  2. 1.5 ml Eppendorf tubes
  3. Millipore 0.22 µm pore size filter (EMD Millipore, catalog number: SLGP033RS )
  4. 9-cm sterile Petri dishes
  5. Electroporation cuvettes 0.2 cm gap (Bio-Rad Laboratories, catalog number: 1652082 )
  6. Glass beads for bacterial plating (VWR, catalog number: 201-0279 )
  7. pKD267 plasmid (described in Maisonneuve et al., 2011)
  8. pKD46 plasmid (described in Datsenko and Wanner, 2000)
  9. Expand High Fidelity DNA polymerase (Roche Molecular Systems, catalog number: 11732641001 )
  10. DpnI restriction enzyme (New England Biolabs, catalog number: R0176 )
  11. PCR purification kit (5Prime, catalog number: 2300610 )
  12. Plasmid purification kit (Roche Molecular Systems, catalog number: 11754777001 )
  13. 4 °C chilled sterile distilled MilliQ water
  14. Distilled MilliQ water
  15. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A0166 )
  16. L-arabinose (Sigma-Aldrich, catalog number: A3256 )
  17. Kanamycin (Sigma-Aldrich, catalog number: K1876 )
  18. D-glucose (Sigma-Aldrich, catalog number: G8270 )
  19. Bacto tryptone (BD, BactoTM, catalog number: 211705 )
  20. Bacto yeast extract (BD, BactoTM, catalog number: 288620 )
  21. Sodium chloride (NaCl) (VWR, BDH®, catalog number: 102415K )
  22. European bacteriological agar (Conda, catalog number: 1800 )
  23. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 71645 )
  24. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P9791 )
  25. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A9434 )
  26. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C3881 )
  27. Magnesium sulfate (MgSO4) (VWR, BDH®, catalog number: 291175X )
  28. Vitamin B1 (Thiamine) (Sigma-Aldrich, catalog number: T4625 )
  29. L-rhamnose monohydrate (Sigma-Aldrich, catalog number: R3875 )
  30. Potassium chloride (KCl)
  31. Na2HPO4·12H2O
  32. Glycerol (v/v) (Sigma-Aldrich, catalog number: G5516 )
  33. Luria-Bertani (LB) broth medium (see Recipes)
  34. Luria Bertani plates (see Recipes)
  35. M9 (10x) (see Recipes)
  36. CaCl2/MgSO4 solution (100x)
  37. M9 minimum medium (see Recipes)
  38. M9-rhamnose plates (see Recipes)
  39. Phosphate saline buffered (PBS) (see Recipes)
  40. 10% sterile glycerol (see Recipes)
  41. L-rhamnose (see Recipes)

Equipment

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

Software

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

Procedure

This procedure is only designed to study the stability of plasmids carried by bacterial species able to uptake rhamnose from the medium, and species in which the ParE toxin is functional. This should be considered before trying to adapt this procedure to new species. Although we employed this assay to measure the stability of the virulence plasmid of Salmonella enterica subs. enterica serovar Typhimurium str. SV5015 (Lobato-Marquez et al., 2016), the method is also useful for E. coli plasmids.

  1. Design of recombinant plasmid variants
    1. Amplify aph-parE cassette by polymerase chain reaction (PCR) using pKD267 plasmid as template and Expand High Fidelity DNA polymerase (~1,800 bp) (see Notes 1 and 2, Table 1 and Figure 1). It is recommended to use the following PCR program: 94 °C 5 min, [94 °C 30 sec; 60 °C 30 sec; 72 °C 2 min] x 10, [94 °C 30 sec; 60 °C 30 sec; 72 °C 2 min + 5 sec increment per cycle] x 25 cycles.
    2. Digest the PCR-amplified aph-parE cassette by adding 1 µl of DpnI per 50 µl of PCR reaction. DpnI digests methylated DNA, thus eliminating the parental pKD267 plasmid but not the amplified aph-parE DNA module.
    3. Purify the DpnI-digested PCR reaction using a 5Prime PCR purification kit. Elute DNA in MilliQ water. Alternatively, standard precipitation procedures such as the phenol/chloroform precipitation method, can be used to purify the aph-parE DNA fragment (see Note 3).
    4. Concentrate purified-PCR product to 500-800 ng/µl using DNA SpeedVac.
    5. Transform the strain containing the plasmid for which the stability is to be analyzed, with pKD46 (see Note 4 and Figure 3). Grow the resulting variant in LB containing 50 µg/ml of ampicillin at 30 °C.
    6. Make electrocompetent cells of the strain carrying pKD46 (Figure 3). Pick one colony of the new strain carrying pKD46 plasmid and inoculate 5 ml of LB supplemented with 50 µg/ml of ampicillin and 0.4% arabinose (w/v). Grow the culture for 16 h at 30 °C in 10 ml LB in a 100 ml flask with shaking (~150 rpm).
      1. Dilute 1:100 of overnight culture in LB containing 50 µg/ml of ampicillin and 0.4% arabinose in 10:1 flask:medium volume ratio flask.
      2. Grow the bacterial culture at 30 °C and 150 rpm up to an optical density (measured at 600 nm) of 0.6.
      3. Centrifuge the bacterial culture in a 4 °C refrigerated centrifuge for 5 min at 15,557 x g.
      4. Discard the supernatant and rinse the bacterial pellet twice with 4 °C chilled sterile distilled water.
      5. Discard the supernatant and rinse the bacterial pellet with 4 °C sterile 10% glycerol.
      6. Resuspend the bacterial pellet in sterile 10% glycerol (500 µl per 50 ml of bacterial culture).
      7. Aliquot bacterial electrocompetent cells in 1.5 ml Eppendorf tubes (250 µl culture per tube). Keep on ice if competent cells are going to be used immediately. Otherwise, electrocompetent cells should be kept at -80 °C.
    7. Electroporate pKD46 competent cells (2.5 kV, 5 ms) with 800-1,000 ng of purified aph-parE DNA fragment (Figure 3) and grow cells for 3 h at 37 °C (this temperature promotes the loss of pKD46).
    8. Plate electroporated cells in LB-plates containing 50 µg/ml of kanamycin and 0.2% of glucose (see Note 5).
    9. Confirm the proper integration of the aph-parE cassette in your plasmid using PCR.


      Figure 3. Scheme summarizing the design of recombinant plasmid variants

  2. Plasmid stability assay
    Note: See Note 6 before starting the assay.
    1. Inoculate the bacterial culture in 10 ml of LB without selective pressure in a 100 ml flask (10:1 flask:medium volume ratio) at 37 °C and 150 rpm. Always adjust the amount of inoculum of all used strains by measuring the optical density (600 nm). Grow cultures according to the number of desired bacterial generations (see Notes 7 and 8). For instance, this protocol was optimized for Salmonella enterica subsp. enterica serovar Typhimurium grown for 16 h at 37 °C (~10 generations).
    2. Collect 1 ml of grown cultures in 1.5 ml Eppendorf tubes.
    3. Centrifuge for 1 min at ~10,800 x g at room temperature using a benchtop centrifuge (MiniSpin® Eppendorf), or an equivalent speed in a different benchtop centrifuge.
    4. Discard supernatants and wash bacterial pellets twice with 1 ml of phosphate buffered saline (PBS) pH 7.4 (see Note 9). Resuspend bacterial pellets in 1 ml PBS.
    5. Perform 1:10 serial dilutions using 100 µl of PBS-resuspended bacterial cultures in PBS-containing Eppendorf tubes.
    6. Plate 100 µl of the appropriate dilutions onto LB-plates and M9-rhamnose plates by using glass beads. Proper dilution must be adjusted according to the studied plasmid (see Notes 10 and 11).
    7. Incubate plates for 24 h (LB-agar) or 48-72 h (M9-rhamnose-agar) at 37 °C before counting the number of colony forming units (CFUs) (see Note 12).
    8. To discard false positives, CFUs grown in M9-rhamnose-agar should be tested for their kanamycin resistance by streaking them onto antibiotic-containing LB plates.

Data analysis

  1. Count the number of CFUs in M9-rhamnose and in LB plates
  2. The segregants fraction (the fraction of bacteria that have lost the studied plasmid) can be calculated as the ‘number CFUs per ml in M9-rhamnose’/‘number CFUs per ml in LB’ (Lobato-Marquez et al., 2016).
  3. Analyze stability differences between strains by t-test or one-way ANOVA. If ANOVA is used post hoc tests can be employed to determine mean differences between all strains (Tukey’s test) or to compare test strains to the control strain (Dunnett’s test). We suggest using GraphPad Prism software (La Jolla, USA)

Notes

  1. This stability assay is based on the insertion of the aph-parE cassette in the plasmid/gene of interest. The cassette integration procedure is adapted from the original method described by Datsenko and Wanner (2000) and summarized in Figure 1.
  2. Primer design and aph-parE amplification: design oligonucleotides that bind to the aph-parE region of pKD267 plasmid (Table 1). Primers must contain 50 bp homologous to the upstream (forward) and downstream (reverse) regions of the desired insertion site, in the 5’-end of the sequence annealing with pKD267 (Figure 1, Table 1). To test the stability of the plasmid of interest, the aph-parE cassette must be inserted into a region of the plasmid which is not involved in plasmid stability. If testing the contribution of a specific gene to plasmid stability, the aph-parE cassette must be inserted in such a way that the gene of interest is disrupted (preferentially also inactivating the promoter of that gene, to avoid polar effects).

    Table 1. DNA sequence of the 3’-end oligonucleotides annealing with the pKD267 aph-parE cassette
    Primer name
    Primer sequence (5’-3’)
    Used for
    Forward 3’-end
    TCTCTACGCCGGACGCATCGTG
    Amplify aph-parE cassette
    Reverse 3’-end
    ACTGATCAGTGATAAGCTGTC
    Amplify aph-parE cassette
    Km-Comp5
    CACGATGCGTCCGGCGTAGAG
    Check aph-parE insertions
    Km-Comp3
    GACAGCTTATCACTGATCAG
    Check aph-parE insertions

    When designing the forward 3’-end and reverse 3’-end primers, it is necessary to add the 50 bp homologous to the insertion site (see Figure 1).
  3. When purifying the aph-parE cassette it is important to remove as much of the salts as possible to prevent interferences with subsequent electroporation.
  4. The pKD46 plasmid is a thermo-sensitive plasmid encoding λ-Red recombinase necessary to integrate the aph-parE cassette into the desired region of the plasmid. λ-Red recombinase synthesis is induced in the presence of arabinose. Once transformed with pKD46 plasmid, the resulting recombinant strain must be grown at 30 °C and in the presence of 50 µg/ml of ampicillin until electroporation with the aph-parE fragment.
  5. After insertion of aph-parE cassette into the plasmid of interest it is highly recommended to always grow cells in the presence of 0.2% glucose. Glucose represses the PparE promoter and so avoids any possible toxicity derived from ParE synthesis.
  6. Stability assay: always grow bacteria in the presence of 50 µg/ml of kanamycin before counting the number of desired generations. This will ensure starting the experiment begins with ~100% bacteria-containing plasmid.
  7. If several strains containing different plasmid derivatives are being compared, the researcher should confirm that the growth rate of all strains is the same. Different growth rates mean a different number of bacterial generations and would therefore produce differences in the fraction of segregants.
  8. This methodology was used to measure plasmid stability of pSLT plasmid after ~10 generations and was able to detect ~1 segregant in 2 x 106 bacteria (Lobato-Marquez et al., 2016). Due to the high sensitivity of the described assay we strongly believe that this procedure might be adapted to identify plasmid-free cells in a fewer number of generations.
  9. After growing bacterial cultures under selected conditions, culture aliquots must be washed twice with 1 ml of PBS to remove traces of LB medium. M9-rhamnose-agar plates must contain rhamnose as the only carbon source. The rhamnose promoter is subjected to catabolic repression, meaning that other carbon sources such as glucose repress the expression of parE and therefore prevent the negative selection of plasmid-free cells.
  10. PBS, LB medium and M9-rhamnose plates must be kept sterile. If bacterial cultures containing aph-parE cassette are contaminated with a non aph-parE-containing strain it will produce false positive colonies in M9-rhamnose plates also sensitive to kanamycin.
  11. Working dilutions: as an example, for the highly stable pSLT virulence plasmid of S. typhimurium (~10-7 segregants per cell generation), 1:107 dilution was used to quantify total bacterial population in LB-agar plates, and dilutions in the range 1:1-103 were used to determine the number of segregants in M9-rhamnose-agar plates.
  12. M9-rhamnose plates should not be kept for more than 72 h at 37 °C, except for those bacteria with an extremely low duplication rate. We have observed that ParE toxin activity is more bacteriostatic than bactericidal, meaning that after a long time at 37 °C incubation plasmid-containing cells will grow.

Recipes

  1. Luria Bertani (LB) broth
    10 g/L Bacto tryptone
    5 g/L Bacto yeast extract
    10 g/L NaCl
    Sterilize by autoclaving at 120 °C for 20 min
  2. Luria Bertani plates
    LB medium
    15 g/L agar
    Sterilize by autoclaving at 120 °C for 20 min
  3. M9 (10x)
    176.5 g/L Na2HPO4
    30 g/L KH2PO4
    5 g/L NaCl
    10 g/L NH4Cl
    Filter sterilize
  4. CaCl2/MgSO4 solution (100x)
    0.01 M CaCl2
    0.1 M MgSO4
    Sterilize by filtering using a 0.22 µm pore Millipore filter or autoclaving at 120 °C for 20 min
  5. M9 minimal medium
    1x M9
    1x CaCl2/MgSO4 solution
    1 mg/ml Vitamine B1 (thiamine)
    0.5% rhamnose
    Sterilize water by autoclaving and add the rest of the sterile components. Do not autoclave the final solution. This solution can be kept at room temperature but should be kept protected from light to preserve thiamine integrity
  6. M9-rhamnose plates
    M9 minimal medium
    15 g/L agar
    Sterilize water containing agar by autoclaving at 120 °C for 20 min, and add the rest of the sterile components
  7. Phosphate saline buffered (PBS)
    8 g/L NaCl
    0.2 g/L KCl
    2.89 g/L Na2HPO4·12H2O
    0.2 g/L KH2PO4
    Adjust pH to 7.4
  8. 10% sterile glycerol
    Dilute glycerol in MilliQ water to a final concentration of 10% (v/v)
    Sterilize by autoclaving at 120 °C for 20 min
  9. L-rhamnose
    To prepare a sterile rhamnose solution (we recommend preparing a 15% stock solution), dilute rhamnose in MilliQ water and filter using a 0.22 µm pore Millipore filter

Acknowledgments

This work was performed in the laboratories of Prof. Díaz-Orejas and Prof. García-del Portillo and supported by grants BFU2011-25939, CSD2008-00013, and BIO2013-46281-P/BIO2015-69085-REDC from the Spanish Ministry of Economy and Competitiveness.
We thank Alexandra Willis for her critical review of the manuscript.
This protocol was originally described in Lobato-Márquez et al., 2016.

References

  1. Bochner, B. R., Huang, H. C., Schieven, G. L. and Ames, B. N. (1980). Positive selection for loss of tetracycline resistance. J Bacteriol 143(2): 926-933.
  2. Datsenko, K. A. and Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97(12): 6640-6645.
  3. del Solar, G. H., Puyet, A. and Espinosa, M. (1987). Initiation signals for the conversion of single stranded to double stranded DNA forms in the streptococcal plasmid pLS1. Nucleic Acids Res 15(14): 5561-5580.
  4. Garcia-Quintanilla, M., Prieto, A. I., Barnes, L., Ramos-Morales, F. and Casadesus, J. (2006). Bile-induced curing of the virulence plasmid in Salmonella enterica serovar Typhimurium. J Bacteriol 188(22): 7963-7965.
  5. Gerdes, K., Larsen, J. E. and Molin, S. (1985). Stable inheritance of plasmid R1 requires two different loci. J Bacteriol 161(1): 292-298.
  6. Jiang, Y., Pogliano, J., Helinski, D. R. and Konieczny, I. (2002). ParE toxin encoded by the broad-host-range plasmid RK2 is an inhibitor of Escherichia coli gyrase. Mol Microbiol 44(4): 971-979.
  7. Li, X. T., Thomason, L. C., Sawitzke, J. A., Costantino, N. and Court, D. L. (2013). Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli. Nucleic Acids Res 41(22): e204.
  8. Lobato-Marquez, D., Molina-Garcia, L., Moreno-Cordoba, I., Garcia-Del Portillo, F. and Diaz-Orejas, R. (2016). Stabilization of the virulence plasmid pSLT of Salmonella Typhimurium by three maintenance systems and its evaluation by using a new stability test. Front Mol Biosci 3: 66.
  9. Maisonneuve, E., Shakespeare, L. J., Jorgensen, M. G. and Gerdes, K. (2011). Bacterial persistence by RNA endonucleases. Proc Natl Acad Sci U S A 108(32): 13206-13211.
  10. Maloy, S. R. and Nunn, W. D. (1981). Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol 145(2): 1110-1111.

简介

可以通过阳性选择使用抗生素抗性质粒衍生物来测量质粒稳定性。然而,高度稳定的质粒低于这些测定的灵敏度范围。为了解决这个问题,我们描述了一种新颖的,高度灵敏的方法来测量质粒稳定性,这是基于在含有质粒的细胞消除后,无质粒细胞的选择。这里提出的检测方法是基于aph-parE 盒式磁带。当细胞合成时,ParE毒素诱导细胞死亡。 ParE合成由鼠李糖诱导型启动子控制。当携带 aph-parE 模块的细菌生长在含有鼠李糖作为唯一碳源的培养基中时,合成ParE,消除含有质粒的细胞。进一步使用卡那霉素抗性( aph )来证实在鼠李糖生长的细菌中不存在质粒。

背景 通过使用抗生素抗性质粒衍生物的阳性选择测定质粒稳定性。携带研究质粒的​​细胞在选择性抗生素(Gerdes等人,1985; del Solar等人,1987)的存在下被阳性选择。这种技术的主要缺点是其灵敏度;高度稳定的质粒低于这些测定的敏感性。为了解决这个问题,已经描述了依靠直接选择无质粒细胞例如 - 四环素系统的替代方法(Bochner等人,1980; Maloy和Nunn,1981; Garcia-Quintanilla 等人,2006)。 - 四环素方法的限制包括差的再现性和频繁发生的假阳性(Li等等,2013)。在这里,我们描述了基于含有质粒的细胞的反选择的新颖的,高度灵敏的质粒稳定性测定。该测定基于含有由鼠李糖诱导型启动子和卡那霉素抗性基因(图1)控制的ParE毒素编码基因的盒(图1)(Maisonneuve等人,2011)。 ParE是毒素 - 抗毒素系统parDE的毒素,靶向DNA回旋酶,阻断DNA复制并诱导导致细胞死亡的DNA断裂(Jiang et al。,2002) )。使用同源重组将 aph-parE 盒插入感兴趣的质粒。在含有鼠李糖作为唯一碳源的基本培养基中诱导PparE时,只有无质粒的细胞才能存活(Lobato-Marquez等人,2016)。然后使用卡那霉素确认质粒的损失(图2)。


图1.示出了将 aph-parE 盒整合到感兴趣的质粒中的方案。(1) 首先通过PCR使用pKD267质粒作为模板进行扩增。 (2)然后,携带编码λ-Red重组酶的质粒的细胞用aph-parE DNA片段电穿孔。 λ-Red重组酶将 aph-parE 模块的特异性整合引导到包含用于PCR的寡核苷酸中包含的50bp上游和50bp下游同源序列的质粒区。 (3)通过使用引物退火盒(红色箭头)和质粒(黑色箭头)来确认插入片段(aph-parE )。


图2.质粒稳定性程序为了避免质粒损失,携带 aph-parE盒的菌株最初在抗生素选择压力下生长(使用卡那霉素)。当从培养基中取出卡那霉素时,感兴趣的质粒将在一定数量的代数之后丢失。当将培养物接种在含有鼠李糖作为唯一碳源的M9-鼠李糖板中时,选择无质粒细胞。从Lobato-Marquez等人修改,2016年。

关键字:质粒稳定性, ParE, 毒素, 负向选择, 革兰氏阴性菌

材料和试剂

  1. 移液器提示
  2. 1.5 ml Eppendorf管
  3. Millipore0.22μm孔径过滤器(EMD Millipore,目录号:SLGP033RS)
  4. 9厘米无菌培养皿
  5. 电穿孔试管0.2厘米间隙(Bio-Rad实验室,目录号:1652082)
  6. 用于细菌电镀的玻璃珠(VWR,目录号:201-0279)
  7. pKD267质粒(描述于Maisonneuve等人,2011)
  8. pKD46质粒(在Datsenko和Wanner,2000中描述)
  9. 展开高保真DNA聚合酶(Roche Molecular Systems,目录号:11732641001)
  10. IpI限制酶(New England Biolabs,目录号:R0176)
  11. PCR纯化试剂盒(5Prime,目录号:2300610)
  12. 质粒纯化试剂盒(Roche Molecular Systems,目录号:11754777001)
  13. 4℃冷冻无菌蒸馏MilliQ水
  14. 蒸馏MilliQ水
  15. 氨苄西林钠盐(Sigma-Aldrich,目录号:A0166)
  16. L-阿拉伯糖(Sigma-Aldrich,目录号:A3256)
  17. 卡那霉素(Sigma-Aldrich,目录号:K1876)
  18. D-葡萄糖(Sigma-Aldrich,目录号:G8270)
  19. Bacto胰蛋白胨(BD,Bacto TM ,目录号:211705)
  20. 细菌酵母提取物(BD,Bacto TM,目录号:288620)
  21. 氯化钠(NaCl)(VWR,BDH ,目录号:102415K)
  22. 欧洲细菌琼脂(Conda,目录号:1800)
  23. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:71645)
  24. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P9791)
  25. 氯化铵(NH 4 Cl)(Sigma-Aldrich,目录号:A9434)
  26. 氯化钙二水合物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C3881)
  27. 硫酸镁(MgSO 4)(VWR,BDH,目录号:291175X)
  28. 维生素B1(硫胺素)(Sigma-Aldrich,目录号:T4625)
  29. L-鼠李糖一水合物(Sigma-Aldrich,目录号:R3875)
  30. 氯化钾(KCl)
  31. Na 2 HPO 4/12H 2 O
  32. 甘油(v/v)(Sigma-Aldrich,目录号:G5516)
  33. Luria-Bertani(LB)肉汤培养基(见食谱)
  34. Luria Bertani盘(见食谱)
  35. M9(10x)(见配方)
  36. CaCl 2/MgSO 4溶液(100x)
  37. M9最低介质(见配方)
  38. M9-鼠李糖板(参见食谱)
  39. 磷酸盐缓冲液(PBS)(见食谱)
  40. 10%无菌甘油(见食谱)
  41. L-鼠李糖(见食谱)

设备

  1. 选择单道移液管(2μl,20μl,20μl,1,000μl)(Gilson,P-2,P-20,P-200,P-1000)
  2. 玻璃瓶100ml(Fisher Scientific,Fisherbrand)
  3. 玻璃50ml烧瓶(Fisher Scientific,Fisherbrand)
  4. MiniSpin ® Eppendorf台式离心机(Eppendorf,型号:MiniSpin ®
  5. 细菌培养箱(Eppendorf,New Brunswick,型号:Innova 4000)
  6. MicroPulser TM 电穿孔机(Bio-Rad Laboratories,型号:MicroPulser Electroporator,目录号:1652100)
  7. Eppendorf冷冻离心机(Eppendorf,型号:5804)
  8. 分光光度计(Thermo Fisher Scientific,Thermo Scientific TM ,型号:SPECTRONIC TM 200)
  9. Milli-Q ®超纯水(Nanopure)水净化系统
  10. DNA SpeedVac(Savant Systems,型号:SpeedVac DNA 110)
  11. 高压灭菌器(Prestige Medical,目录号:210004)

软件

  1. GraphPad Prism软件( https://www.graphpad.com/scientific -software /棱镜/

程序

该程序仅用于研究能够从培养基中吸收鼠李糖的细菌物种和ParE毒素功能的物种携带的质粒的稳定性。在尝试将这一程序适应新物种之前,应该考虑这一点。尽管我们采用这种测定来测量肠炎沙门氏菌的毒力质粒的稳定性。肠毒素 serovar Typhimurium str。 SV5015(Lobato-Marquez等人,2016),该方法对于E也是有用的。大肠杆菌质粒。

  1. 重组质粒变体的设计
    1. 使用pKD267质粒作为模板并扩增高保真DNA聚合酶(〜1,800bp),通过聚合酶链反应(PCR)扩增aph-parE 盒(参见注释1和2,表1和图1)。建议使用以下PCR程序:94℃5分钟,[94℃30秒; 60℃30秒; 72℃2分]×10,[94℃30秒; 60℃30秒; 72°C每周期2分+ 5秒增量)×25次循环
    2. 通过每50μlPCR反应加入1μl的DpnI,对PCR扩增的aph-parE 盒进行消化。 Dpn我消化甲基化DNA,从而消除了亲本pKD267质粒,而不是扩增的aph-parE DNA模块。
    3. 使用5Prime PCR纯化试剂盒纯化I-digested PCR反应的 Dpn。在MilliQ水中洗脱DNA。或者,可以使用诸如苯酚/氯仿沉淀法的标准沉淀方法来纯化DNA片段(参见附注3)。
    4. 使用DNA SpeedVac将纯化的PCR产物浓缩至500-800ng /μl。
    5. 用pKD46转化含有待分析稳定性的质粒的菌株(见附注4和图3)。在30℃下生长含有50μg/ml氨苄青霉素的LB的变体
    6. 制备携带pKD46的菌株的电感受态细胞(图3)。挑取携带pKD46质粒的新菌株的一个菌落,并接种5ml补充有50μg/ml氨苄青霉素和0.4%阿拉伯糖(w/v)的LB。在100毫升摇瓶(约150转/分钟)的烧瓶中,将培养物在30℃下在10ml LB培养物中培养16小时。
      1. 在10:1烧瓶:中等体积比例的烧瓶中稀释1:100的含有50μg/ml氨苄青霉素和0.4%阿拉伯糖的LB的过夜培养物。
      2. 在30℃和150rpm下培养细菌培养物,达到0.6的光密度(600nm测量)。
      3. 将细菌培养物在4℃冷冻离心机中以15,557×g离心5分钟。
      4. 弃去上清液,用4℃冷冻无菌蒸馏水冲洗细菌沉淀两次
      5. 弃去上清液,用4℃无菌10%甘油冲洗细菌沉淀
      6. 将细菌沉淀重悬于无菌的10%甘油(每50ml细菌培养物500μl)中
      7. 在1.5ml Eppendorf管中分装细菌电感受态细胞(每管250μl培养物)。如果有能力的细胞将立即使用,请保持在冰上。否则,电感应细胞应保持在-80°C
    7. 将电穿孔pKD46感受态细胞(2.5kV,5ms)与800-1,000ng纯化的aph-parE DNA片段(图3)连接,并在37℃下培养细胞3小时(该温度促进损失的pKD46)
    8. 在含有50μg/ml卡那霉素和0.2%葡萄糖的LB平板中的板电穿孔细胞(参见注释5)。
    9. 使用PCR确认您的质粒中 aph-parE 盒的正确整合。


      图3.总结重组质粒变体设计的方案

  2. 质粒稳定性分析
    注意:在开始测定之前,请参阅注释6.
    1. 在37℃和150rpm下,在100ml烧瓶(10:1烧瓶:中等体积比)中将10ml无LB的细菌培养物接种在无选择压力下。通过测量光密度(600 nm),始终调整所有使用菌株的接种量。根据所需的细菌代数培养培养物(见注释7和8)。例如,该协议针对肠炎沙门菌子项进行了优化。肠杆菌血清型鼠伤寒沙门氏菌在37℃(〜10代)生长16小时。
    2. 在1.5 ml Eppendorf管中收集1 ml生长的培养物。
    3. 使用台式离心机(MiniSpin,Eppendorf)在室温下以约10,800 x g的速度离心1分钟,或在不同台式离心机中等效的速度。
    4. 丢弃上清液,并用1 ml磷酸盐缓冲盐水(PBS)pH 7.4(见注9)清洗细菌沉淀两次。将细菌沉淀重悬于1 ml PBS中
    5. 使用100μlPBS重悬细菌培养物在含PBS的Eppendorf管中进行1:10连续稀释。
    6. 通过使用玻璃珠将100μl适当的稀释液平板放置在LB平板和M9-鼠李糖板上。必须根据研究的质粒调整适当的稀释度(见注释10和11)。
    7. 在计数菌落形成单位数(CFU)之前,在37℃下培养板24小时(LB-琼脂)或48-72小时(M9-鼠李糖琼脂)(见附注12)。
    8. 为了放弃假阳性,应在M9-鼠李糖琼脂中生长的CFU通过将其贴在含抗生素的LB平板上来测试他们的卡那霉素抗性。

数据分析

  1. 计算M9-鼠李糖和LB板中的CFU数量
  2. 分离子级分(已经失去研究的质粒的细菌的分数)可以计算为LB中的"M9-鼠李糖"/'CFU/ml中的每毫升数的CFU(Lobato-Marquez等人, em>。,2016)。
  3. 通过测试或单因素方差分析分析菌株之间的稳定性差异。如果使用方差分析,则可以采用事后检验来确定所有菌株(Tukey's检验)之间的平均差异,或者将检测菌株与对照菌株(Dunnett's检验)进行比较。我们建议使用GraphPad Prism软件(La Jolla,USA)

笔记

  1. 该稳定性测定基于将 aph-parE 盒插入感兴趣的质粒/基因。盒式集成程序根据Datsenko和Wanner(2000)描述的原始方法进行了调整,并在图1中进行了总结。
  2. 引物设计和aph-parE扩增:结合pKD267质粒的 aph-parE 区域的设计寡核苷酸(表1)。在pKD267序列退火的5'-末端,引物必须含有与所需插入位点的上游(正向)和下游(反向)区域同源的50bp(图1,表1)。为了测试感兴趣的质粒的稳定性,必须将 aph-parE 盒插入质粒的不涉及质粒稳定性的区域。如果测试特定基因对质粒稳定性的贡献,则必须以这样的方式插入目标基因被破坏(优先也使该基因的启动子失活以避免极地效应)
    表1.使用pKD267 入门名称
    引物序列(5'-3')
    用于
    正向3'末端
    TCTCTACGCCGGACGCATCGTG
    放大aph-parE 磁带盒
    反向3'末端
    ACTGATCAGTGATAAGCTGTC
    放大aph-parE 磁带盒
    Km-Comp5
    CACGATGCGTCCGGCGTAGAG
    检查aph-parE 插入
    Km-Comp3
    GACAGCTTATCACTGATCAG
    检查aph-parE 插入

    当设计正向3'末端和反向3'端引物时,有必要将50bp同源于插入位点(见图1)。
  3. 当纯化 aph-parE 盒时,重要的是尽可能多地除去盐,以防止随后的电穿孔产生干扰。
  4. pKD46质粒是编码λ-Red重组酶的热敏质粒,其将 aph-parE 盒整合到质粒的所需区域中。在阿拉伯糖存在下诱导λ-Red重组酶合成。一旦用pKD46质粒转化,所得到的重组菌株必须在30℃和50μg/ml氨苄青霉素存在下生长,直到用aph-parE片段进行电穿孔。
  5. 在将 aph-parE 盒插入感兴趣的质粒后,强烈建议在0.2%葡萄糖存在下总是生长细胞。葡萄糖抑制 启动子,因此避免由ParE合成引起的任何可能的毒性。
  6. 稳定性测定:在计算所需代数之前,总是在存在50μg/ml卡那霉素的情况下生长细菌。这将确保开始实验,开始含〜100%含细菌的质粒。
  7. 如果比较含有不同质粒衍生物的几种菌株,研究人员应确认所有菌株的生长速率是相同的。不同的生长速率意味着不同数量的细菌代,因此会产生分离子分数的差异。
  8. 该方法用于测量〜10代后pSLT质粒的质粒稳定性,并且能够检测2×10 6个细菌中的〜1个分离物(Lobato-Marquez等人)。 ,2016)。由于所述测定的高灵敏度,我们强烈认为,该过程可能适应于以较少代数识别无质粒细胞。
  9. 在选择的条件下培养细菌培养物后,培养液必须用1ml PBS洗涤两次以除去痕量的LB培养基。 M9-鼠李糖琼脂平板必须含有鼠李糖作为唯一的碳源。鼠李糖启动子经受分解代谢抑制,意味着其他碳源(如葡萄糖)抑制了parE的表达,因此可以防止无质粒细胞的阴性选择。
  10. PBS,LB培养基和M9-鼠李糖板必须保持无菌。如果含有 aph-parE 盒的细菌培养物被含有不含aph-parE的菌株污染,则在对卡那霉素也敏感的M9-鼠李糖板中将产生假阳性菌落。 br />
  11. 工作稀释液:作为例子,对于高稳定性的pSLT毒力质粒。使用1:10 <7>稀释法定量LB-琼脂平板上的总细菌总数,稀释液使用范围1:1-10 3 来确定M9-鼠李糖琼脂平板上的分离物数量。
  12. M9-鼠李糖板不应在37℃下保存72小时以上,重复率极低的细菌除外。我们已经观察到,ParE毒素活性比杀菌剂更具杀菌性,这意味着在37℃下长时间培养含有质粒的细胞将会生长。

食谱

  1. Luria Bertani(LB)肉汤
    10克/升Bacto胰蛋白胨
    5 g/L Bacto酵母提取物
    10g/L NaCl
    用120℃高压消毒灭菌20分钟
  2. Luria Bertani盘子
    LB培养基
    15 g/L琼脂
    用120℃高压消毒灭菌20分钟
  3. M9(10x)
    176.5g/L Na 2 HPO 4
    30g/L KH PO 4
    5克/升NaCl
    10g/L NH 4 Cl
    过滤灭菌
  4. CaCl 2/MgSO 4溶液(100x)
    0.01M CaCl 2
    0.1M MgSO 4
    通过使用0.22微米孔微孔过滤器过滤或在120℃下高压灭菌20分钟进行消毒
  5. M9极限媒体
    1x M9
    1×CaCl 2/MgSO 4 溶液
    1 mg/ml维生素B1(硫胺素)
    0.5%鼠李糖 通过高压灭菌消毒水并加入其余的无菌成分。不要对最终解决方案进行高压灭菌。该溶液可以保持在室温,但应保持光线不变,以维持硫胺素完整性
  6. M9-鼠李糖板
    M9极限媒体
    15 g/L琼脂
    通过在120℃高压灭菌20分钟灭菌含有琼脂的水,并加入其余的无菌成分。
  7. 磷酸盐缓冲液(PBS)
    8克/升NaCl
    0.2克/升KCl
    2.89g/L Na 2 HPO 4/12H 2 O
    0.2g/L KH 2 PO 4
    将pH调节至7.4
  8. 10%无菌甘油
    在MilliQ水中稀释甘油至10%(v/v)的最终浓度 用120℃高压消毒灭菌20分钟
  9. L-鼠李糖
    为了制备无菌的鼠李糖溶液(我们建议制备15%的储备溶液),用MilliQ水稀释鼠李糖,并使用0.22μm孔的Millipore过滤器过滤

致谢

这项工作是在Díaz-Orejas教授和García-del Portillo教授的实验室进行的,由西班牙经济部拨款BFU2011-25939,CSD2008-00013和BIO2013-46281-P/BIO2015-69085-REDC支持和竞争力 感谢亚历山德拉·威利斯对稿件的批判性评论 该协议最初在Lobato-Márquez等人,2016中描述。

参考

  1. Bochner,BR,Huang,HC,Schieven,GL和Ames,BN(1980)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/6259126 "target ="_ blank">四环素抗性丧失的正面选择。细菌学 143(2):926-933。
  2. Datsenko,KA和Wanner,BL(2000)。  One - 使用PCR产物使大肠杆菌K-12中的染色体基因失活。 Proc Natl Acad Sci USA 97(12):6640-6645。 />
  3. del Solar,GH,Puyet,A.and Espinosa,M。(1987)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/3039461" target ="_ blank">在链球菌质粒pLS1中单链和双链DNA形式的转化的启动信号。核酸Res 15(14):5561-5580。 />
  4. Garcia-Quintanilla,M.,Prieto,AI,Barnes,L.,Ramos-Morales,F.and Casadesus,J。(2006)。  肠杆菌诱导的肠炎沙门氏菌血清型鼠伤寒杆菌中的毒力质粒的固化。/em> 188(22):7963-7965。
  5. Gerdes,K.,Larsen,JE和Molin,S。(1985)。质粒R1的稳定遗传需要两个不同的基因座。细菌161(1):292-298。
  6. Jiang,Y.,Pogliano,J.,Helinski,DR和Konieczny,I。(2002)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/12010492"target ="_ blank">由广泛宿主范围质粒RK2编码的ParE毒素是大肠杆菌促旋酶的抑制剂.Mol Microbiol 44(4):971-979。
  7. Li,XT,Thomason,LC,Sawitzke,JA,Costantino,N。和Court,DL(2013)。  使用tetA-sacB盒的阳性和阴性选择:大肠杆菌中的重组和P1转导。 em> 41(22):e204。
  8. Lobato-Marquez,D.,Molina-Garcia,L.,Moreno-Cordoba,I. Garcia-Del Portillo,F.and Diaz-Orejas,R。(2016)。  三个维护系统稳定沙门氏菌毒力的毒力质粒pSLT及其评估通过使用新的稳定性测试。前面的Mol Biosci 3:66.
  9. Maisonneuve,E.,莎士比亚,LJ,Jorgensen,MG和Gerdes,K.(2011)。 RNA内切核酸酶的细菌持久性。 Proc Natl Acad Sci USA 108(32):13206-13211。
  10. Maloy,SR和Nunn,WD(1981)。选择 通过大肠杆菌损失四环素抗性。 J Bacteriol 145(2):1110-1111。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
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.
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