参见作者原研究论文

本实验方案简略版
Oct 2017
Advertisement

本文章节


 

Induction of Natural Competence in Genetically-modified Lactococcus lactis
转基因乳酸乳球菌自然感受态的诱导   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Natural competence can be activated in Lactoccocus lactis subsp lactis and cremoris upon overexpression of ComX, a master regulator of bacterial competence. Herein, we demonstrate a method to activate bacterial competence by regulating the expression of the comX gene by using a nisin-inducible promoter in an L. lactis strain harboring either a chromosomal or plasmid-encoded copy of nisRK. Addition of moderate concentrations of the inducer nisin resulted in concomitant moderate levels of ComX, which led to an optimal transformation rate (1.0 x 10-6 transformants/total cell number/g plasmid DNA). Here, a detailed description of the optimized protocol for competence induction is presented.

Keywords: Natural competence (自然感受态), Transformation (转化), Lactococcus lactis (乳酸乳球菌), ComX overexpression (ComX 过表达), NICE system (NICE系统)

Background

Natural competence is the process in which a bacterium acquires exogenous DNA via a specialized uptake machinery after which the internalized DNA is either integrated into its genome or maintained as plasmid DNA. Several bacteria enter a state of competence upon specific environmental triggers such as genotoxic stress or starvation (Seitz and Blokesch, 2013; Blokesch, 2016). Quorum sensing systems such as comCDE or comRS control the activation of natural competence in Gram positive bacteria (Håvarstein et al., 1995; Pestova et al., 1996; Kleerebezem et al., 1997b; Fontaine et al., 2015). More specifically, comC and comS encode pheromones, whereas comD encodes a histidine kinase and comE and comR encode response regulators (Håvarstein et al., 1995; Pestova et al., 1996; Fontaine et al., 2010; Fontaine et al., 2015). In streptococci, the activated regulator drives transcription of the alternative sigma factor ComX, which in turn, activates transcription of competence genes that encode the proteins encompassing the DNA uptake machinery (Johnston et al., 2014). Previously, strategies employing overexpression of ComX led to the successful introduction of exogenous DNA in Streptococcus thermophilus (Blomqvist et al., 2006) and L. lactis (David et al., 2017; Mulder et al., 2017), even though different approaches were employed to achieve its overexpression. These studies also showed that different expression levels of comX critically impact on transformation rates. For example, in our work we used L. lactis subsp. lactis KF147, a strain that allows Nisin-Controlled gene Expression system (NICE) (Mierau and Kleerebezem, 2005) and harbors chromosomal nisRK (essential to allow nisin induction) but does not produce nisin. In this strain, we introduced pNZ6200, a vector containing comX under the control of the nisin-inducible nisA promotor, by electro-transformation, and the resulting strain was not transformable upon full comX induction (2 ng/ml nisin), whereas optimal transformation rates (1.0 x 10-6 transformants/total cell number/g plasmid DNA) were observed upon moderate levels of induction (0.03 ng/ml nisin) (Mulder et al., 2017). Moreover, we also demonstrated that applying the same strategy worked for a nisRK derivative of L. lactis subsp. lactis IL1403, whereas competence induction in L. lactis subsp. cremoris KW2 required prior transformation of the strain with pNZ9531 that expresses nisRK (Kleerebezem et al., 1997a) to allow nisin induced expression of comX. In order to assess whether other L. lactis strains can become naturally competent, we hereby provide a method containing all details of the competence protocol as described previously (Mulder et al., 2017) that can assist other scientists to unleash competence in the L. lactis strain of their interest and might prevent experimental issues concerning nisin induced expression of ComX. This newly developed protocol is expected to allow genetic access in a broad panel of L. lactis strains with an efficiency that enables rapid-one-step construction of gene replacement mutants via integration of linear DNA fragments harboring an antibiotic resistance marker flanked by chromosomal homologous DNA regions.

Materials and Reagents

  1. Pipette tips
  2. 15 ml and 50 ml CELLSTAR® Polypropylene Tube (conical) (Greiner Bio One International, CELLSTAR®, catalog numbers: 188271 and 227261 respectively)
  3. Plastic Petri dish 94 x 16 with vents light version (Greiner Bio One International, catalog number: 633181 )
  4. 12 ml Cell Culture Tubes (Greiner Bio One International, CELLSTAR®, catalog number: 163160 )
  5. Inoculation loops
  6. Eppendorf Tubes® Safe-Lock Tubes 1.5 ml (Eppendorf, catalog number: 0030120086 )
  7. Electroporation cuvettes, 2 mm gap (Bio-Rad Laboratories, catalog number: 1652086 )
  8. Semi-micro cuvettes for 1 ml (for optical density measurements)
  9. Bacterial strains: L. lactis strain of interest with a complete set of competence genes, L. lactis harboring pNZ6200 (Mulder et al., 2017), pNZ6202 (Mulder et al., 2017), and pNZ9531 (Kleerebezem et al., 1997a)
  10. M17 broth (M17) and M17 agar (M17A) (Tritium, catalog numbers: M086.76.0200 and M085.76.0200 respectively)
  11. Glucose solution (20%, Tritium, catalog number: G209.65.0080 )
  12. Distilled water (Thermo Fisher Scientific, GibcoTM, catalog number: 15230089 )
  13. Stock solutions of antibiotics:
    20 mg/ml chloramphenicol (Sigma-Aldrich, catalog number: C0378-100G )
    20 mg/ml erythromycin (Fisher Scientific, catalog number: BP920-25 )
    12.5 mg/ml Tetracycline hydrochloride (Sigma-Aldrich, catalog number: T7660-25G )
  14. NisinA® P Ultrapure Nisin A (Handary, Brussels, Belgium, prepare a 2 mg/ml stock solution in distilled water containing 0.05 glacial acetic acid)
  15. Alternatively: nisin from Lactococcus lactis 2.5% (Sigma-Aldrich, catalog number: N5764 )
  16. JETSTAR 2.0 Maxiprep Kit (GENPRICE, catalog number: 220 020 ) or PureLinkTM HiPure Plasmid Maxiprep Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: K210007 )
  17. QubitTM dsDNA BR Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32850 )
  18. Phenol BioUltra, for molecular biology, TE-saturated, ~73% (T) (Sigma-Aldrich, catalog number: 77607 )
  19. Chloroform HPLC grade, ≥ 99.9% (Sigma-Aldrich, catalog number: 528730 )
  20. Ethidium bromide (Sigma-Aldrich, catalog number: E1510-10ML )
  21. Agarose tablets (U.S. Biotech Sources, catalog number: G01PD-500 )
  22. Glacial acetic acid (Scharlab, catalog number: AC03522500 )
  23. β-glycerophosphate (Disodium salt) (Sigma-Aldrich, catalog number: 50020-500G )
  24. Potassium phosphate dibasic (K2HPO4) (Merck, catalog number: 1.05104.1000 )
  25. Potassium phosphate monobasic (KH2PO4) (Merck, catalog number: 1.04873.1000 )
  26. Na-acetate (Merck, catalog number: 1.06268.1000 )
  27. (NH4)3-citrate (Sigma-Aldrich, catalog number: A1332-500G )
  28. Ascorbic acid (VWR, AnalaR NORMPAPUR®, catalog number: 20150.231 )
  29. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sigma-Aldrich, catalog number: M8054-100G )
  30. Adenine (Sigma-Aldrich, Fluka, catalog number: 01830 )
  31. Guanine (Sigma-Aldrich, catalog number: G11950-100G )
  32. Uracil (Sigma-Aldrich, Fluka, catalog number: 94220 )
  33. Xanthine (Sigma-Aldrich, catalog number: X7375-10G )
  34. Alanine (Sigma-Aldrich, catalog number: A7627-100G )
  35. Arginine (Sigma-Aldrich, catalog number: A5006-500G )
  36. Aspartic acid (Sigma-Aldrich, catalog number: A9256-100G )
  37. Cysteine-HCl (Sigma-Aldrich, catalog number: C1276-250G )
  38. Glutamic acid (Sigma-Aldrich, catalog number: G1251-500G )
  39. Glycine (Merck, catalog number: 1.04201.0250 )
  40. Histidine (Sigma-Aldrich, catalog number: H8000-100G )
  41. Leucine (Sigma-Aldrich, catalog number: L8000-100G )
  42. Lysine (Sigma-Aldrich, catalog number: L5626-100G )
  43. Methionine (Sigma-Aldrich, catalog number: M9625-100G )
  44. Phenylalanine (Sigma-Aldrich, catalog number: P2126-100G )
  45. Proline (Sigma-Aldrich, catalog number: P0380-100G )
  46. Serine (Sigma-Aldrich, catalog number: S4500-100G )
  47. Threonine (Sigma-Aldrich, catalog number: T8625-100G )
  48. Tryptophane (Sigma-Aldrich, catalog number: T0254-100G )
  49. Valine (Sigma-Aldrich, catalog number: V0500-100G )
  50. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670-100G )
  51. Calcium chloride dihydrate (CaCl2·2H2O) (Merck, catalog number: 1.02382.0500 )
  52. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: Z0251-100G )
  53. Cobalt(II) sulfate heptahydrate (CoSO4·7H2O) (Sigma-Aldrich, catalog number: C6768-100G )
  54. Copper (II) sulfate pentahydrate (CuSO4·5H2O) (Scharlab, catalog number: CO01010500 )
  55. Ammonium molybdate tetrahydrate ((NH4)6Mo7O24·4H2O) (Sigma-Aldrich, Fluka, catalog number: 09878 )
  56. Iron(II) chloride tetrahydrate (FeCl2·4H2O) (Sigma-Aldrich, catalog number: 44939-50G )
  57. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 30721-1L )
  58. Iron(III) chloride hexahydrate (FeCl3·6H2O) (Sigma-Aldrich, catalog number: F2877-100G )
  59. p-aminobenzoëic acid (Sigma-Aldrich, catalog number: A9879-100G )
  60. Inosine (Sigma-Aldrich, catalog number: I4125-25G )
  61. Orotic acid (Sigma-Aldrich, catalog number: O2750-100G )
  62. Pyridoxamine-HCl (Sigma-Aldrich, catalog number: P9380-5G )
  63. Thymidine (Sigma-Aldrich, catalog number: T9250-10G )
  64. D-biotin (Sigma-Aldrich, catalog number: B4501-5G )
  65. 6,8-thioctic acid (Sigma-Aldrich, catalog number: T5625-5G )
  66. Pyridoxine-HCl (Sigma-Aldrich, catalog number: P9755-25G )
  67. Folic acid (Sigma-Aldrich, catalog number: F7876-25G )
  68. Nicotinic acid (Sigma-Aldrich, catalog number: N4126-5G )
  69. Ca-(D+)pantothenate (Sigma-Aldrich, catalog number: P5155-100G )
  70. Riboflavin (Sigma-Aldrich, catalog number: R4500-25G )
  71. Thiamin-HCl (Sigma-Aldrich, catalog number: T4625-100G )
  72. Vitamin B12 (Sigma-Aldrich, catalog number: V2876-5G )
  73. Alternatively, PCR product (obtained with KOD hot start DNA polymerase)
    Note: Containing an antibiotic resistance marker flanked by chromosomal fragments for integration obtained by using overlap PCR (Horton et al., 1990) if preferred over plasmid transformation, e.g., for the construction of gene replacement mutants.
  74. Optional: KOD hot start DNA polymerase Master Mix (Merck, Novagen, catalog number: 71842-4 )
  75. GSGM17 (see Recipes)
  76. Washing solution 1 (see Recipes)
  77. Washing solution 2 (see Recipes)
  78. Recovery medium (see Recipes)
  79. CDM (see Recipes)
  80. GCDM (chemically defined medium supplemented with glucose) (see Recipes)
  81. Stock solutions for CDM (see Recipes)
    1. Nucleotide solution
    2. MnCl2·4H2O solution
    3. Amino acid solution
    4. Metal solution
    5. Iron solution
    6. Vitamin solution

Equipment

  1. General pipettes
  2. 200 ml bottle
  3. Water bath (for 30 °C and 55 °C)
  4. Incubation stove at 30 °C
  5. Vortex
  6. Autoclave
  7. Microcentrifuge (Eppendorf® Refrigerated Microcentrifuge) (Eppendorf, model: 5417R )
  8. Centrifuge for 15 and 50 ml tubes (Heraeus Megafuge 1.0R)
  9. Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, InvitrogenTM, mode: Qubit® 2.0 )
  10. Electroporator (Bio-Rad Laboratories, model: GenePulserXcellTM )
  11. Spectrophotometer (Genesys 10 UV Spectrophotometer)
  12. Nanodrop ND1000 spectrophotometer (NanoDrop Technologies) (Thermo Fisher Scientific, model: NanoDropTM 1000 )

Procedure

  1. Plasmid DNA isolation
    1. Culture L. lactis harboring either pNZ6200, pNZ6202, or pNZ9531 in 200 ml M17 supplemented with 1% glucose in a 200 ml bottle containing the appropriate antibiotic (Table 1) at 30 °C without shaking overnight.

      Table 1. Characteristics of plasmids used in this protocol


    2. Pellet cells by centrifugation at 5,000 x g for 15 min at room temperature.
    3. Discard the culture supernatant and freeze the pellets at -20 °C for at least 30 min.
    4. Resuspend the pellets in 10 ml resuspension buffer (manufacturer’s solution from the Maxiprep Kit) containing 40 mg lysozyme.
    5. Incubate for 1.5 h at 55 °C without shaking.
    6. Proceed with the plasmid isolation according to the manufacturer’s protocol from either the JETSTAR 2.0 Maxiprep Kit or PureLinkTM HiPure Plasmid Maxiprep Kit.
    7. Include some extra steps to the manufacturer’s protocol: After centrifugation of the lysates to pellet cell debris, perform phenol-chloroform extraction on the supernatants (phenol: chloroform: supernatant in 1:1:1 volume ratio, mix by inverting the tube) and centrifuge at 5,000 x g for 30 min. Transfer the aqueous phase (upper layer) to a new tube and subject to chloroform extraction (aqueous phase: chloroform 1:1 volume) and subsequent centrifuge at 5,000 x g for 15 min. Load purified fractions on the column from the manufacturer’s kit to allow binding of the DNA and continue the steps as indicated in the manual supplied.
    8. Determine DNA concentration of plasmid DNA by Qubit (1 µg of DNA is required per transformation where the concentration of the plasmid solution is based on Qubit measurement).
    9. Check the quality of the isolated plasmid DNA on a 1% agarose gel.

  2. Electrotransformation of pNZ6200 and pNZ9531 into electrocompetent L. lactis cells (Figure 1)


    Figure 1. Schematic overview for evaluation of competence potential (A) and obtaining an L. lactis strain harboring pNZ6200 and pNZ9531, as well as subsequent induction of competence via nisin induction (B). See the notes in the procedure section for specific cases where we advise modifications to this standard flow scheme.

    1. Prepare 1 L of the electro competence growth medium GSGM17, 500 ml washing solution 1 and 500 ml washing solution 2 (see Recipes).
    2. Culture L. lactis initially overnight in 10 ml GM17 in a 12 ml Cell Culture Tube, incubate at 30 °C without shaking.
    3. The next day, inoculate 5 ml culture into 50 ml GSGM17 and incubate cells overnight at 30 °C, without shaking.
    4. Dilute the overnight culture 1:8 in GSGM17 if the overnight culture reached an OD600 of ≥ 0.7 and grow the culture to an OD600 of 0.2-0.3.
    5. Pellet cells by centrifugation at 6,000 x g for 20 min at 4 °C.
    6. Wash the pellet with 1 volume ice-cold washing solution 1.
    7. Pellet cells by centrifugation at 5,000 x g for 20 min at 4 °C.
    8. Wash the pellet with 0.5 volume ice-cold washing solution 2 and incubate on ice for 15 min.
    9. Pellet cells by centrifugation at 5,000 x g for 20 min at 4 °C.
    10. Wash the pellet with 0.25 volume ice-cold washing solution 1 and pellet cells by centrifugation at 5,000 x g for 20 min at 4 °C.
    11. Resuspend cells in 0.01 volume ice-cold washing solution 1 and aliquot cells per 50 µl.
    12. Use fresh electrocompetent cells immediately for transformation with the desired plasmid.
      Option: Store cells for later use at -80 °C.
    13. Keep electro-cuvettes on ice and prepare recovery medium containing M17 supplemented with 1% glucose, 200 mM MgCl2 and 20 mM CaCl2 and store it on ice as well.
    14. Add 1 µg plasmid to electrocompetent cells and transfer the cell suspension with DNA into the electro-cuvette.
      Note: Do not pipet up and down after addition of plasmid DNA to the electrocompetent cells. Mix cells and plasmid DNA by gently flicking the tube.
    15. Electroporate; settings for the electroporator device: voltage = 2,000 V, capacity = 25 µF, resistance = 200 Ω.
      1. Dry the electro-cuvette with a tissue and tap the electro-cuvette gently onto the bench 5 times to slip down the cells to the bottom of the electro-cuvette.
      2. Put the electro-cuvette in the cuvette holder of the electroporator and apply the electrical pulse.
    16. Add 1 ml of ice-cold recovery medium as soon as possible to the electro-cuvette and put the electro-cuvette on ice again.
    17. Pipet the cells from the electro-cuvette into a 1.5 ml Eppendorf tube and incubate samples in a 30 °C water bath for 1 h without shaking.
    18. Pipet serial dilutions for 100 to 10-6 in recovery medium on selection plates with appropriate antibiotics.
    19. Incubate plates for 2-3 days at 30 °C under non-shaking conditions.
    20. Clean streak positive colonies on a GM17 agar plate supplemented with appropriate antibiotics.
      Note: If co-transformation of pNZ9531 and pNZ6200 is unsuccessful, transform cells first with pNZ9531. Select positive colonies and culture colonies in GM17 supplemented with 10 µg/ml erythromycin. Subsequently, repeat the preparation for electro competence cells and introduce pNZ6200 by electroporation.
    21. The next day, culture single colonies from the clean streak plate in GM17 supplemented with appropriate antibiotics and incubate overnight at 30 °C under non-shaking conditions.
    22. Prepare a 25% glycerol stock and store at -80 °C.

  3. Induction of natural competence in L. lactis
    1. Prepare GCDM (see Recipes) and prepare GM17 agar plates with appropriate antibiotics.
    2. Culture L. lactis harboring pNZ6200 and pNZ9531 separately in 10 ml GCDM supplemented with the appropriate antibiotics (Table 1).
    3. Prepare a 50 ml CELLSTAR® Polypropylene Tube containing 50 ml GCDM.
    4. Incubate cells and the 50 ml CELLSTAR® Polypropylene Tube containing 50 ml GCDM at 30 °C overnight in an incubation stove without shaking.
    5. The next day, add chloramphenicol and erythromycin to the 50 ml CELLSTAR® Polypropylene Tube with 50 ml GCDM.
    6. Add 1 ml of the GCDM in a semi-micro cuvette (= blank sample) by measuring optical density at OD600.
    7. Add 750 µl of the L. lactis harboring pNZ6200 overnight culture to the 50 ml CELLSTAR® Polypropylene Tube tube containing GCDM.
    8. Mix the cell culture by inverting the closed tube.
    9. Pipet 1 ml of the culture into a semi-micro cuvette and incubate cells in the 30 °C water bath non-shaking until the culture reaches an OD600 of 0.3 (after approximately 3-4 h).
    10. Measure the initial OD600 of the culture.
      Note: The OD600 of the culture at t = 0 should be around 0.03. If the OD600 is lower than 0.03; add more culture.
    11. Culture cells until an OD600 of 0.3 (corresponds with approximately 2-3 x 108 cells) under non-shaking conditions.
    12. Prepare calculations and recovery medium (see Recipes, 5 ml recovery medium per transformation).
    13. Transfer 10 ml of cell culture at OD600 of 0.3 into a 12 ml sterile tube.
    14. Repeat this step to obtain a total of 3 aliquoted cultures.
      Note: These cultures will be used for the later steps involving the induction of comX expression with 0 ng/ml (negative control), 0.03 ng/ml (optimal) and 2 ng/ml (fully induced) nisin.
    15. Subsequently, prepare the fresh nisin dilutions by using either NisinA® P Ultrapure Nisin A or nisin from Lactococcus lactis 2.5% (Sigma-Aldrich, 2.5% nisin).
    16. Prepare nisin dilutions in glass-tubes by using distilled water containing 0.05% glacial acetic acid in order to increase the stability of the nisin solutions.
      Note: When using nisin from L. lactis 2.5% (Sigma-Aldrich), make sure you correct for the dilution factor (40x) as only 2.5% of the dry matter is nisin.
    17. Induce the 10 ml culture with nisin. For optimal transformation rates, induce cells with nisin to a final concentration of 0.03 ng/ml nisin.
      Notes:
      1. Optional: as a control, include an uninduced sample and a fully induced sample (2 ng/ml).
      2. L. lactis KF147 harboring pNZ6200 induced with 2 ng/ml nisin will stop growth after 1 h induction but other strains might continue growth at this concentration.
    18. Mix immediately by inverting the tubes 3 times after addition of nisin.
    19. Put 600 µl of the induced culture in an Eppendorf tube.
    20. Add 1 µg pNZ6202.
      Note: It is likely that other compatible plasmids can also be used to assess transformation efficiencies.
    21. Mix by inverting the tube.
    22. Incubate at 30 °C for 2 h in a water bath without shaking.
      Note: Longer incubation with nisin does not lead to increased transformation rates (see Figure 2).


      Figure 2. Transformation rate (transformants/total cell number after induction/µg plasmid DNA) after prolonged nisin induction in L. lactis KF147 harboring pNZ6200. Transformation was examined in uninduced (closed circles), moderately induced (closed squares) and fully induced (closed triangles) cultures. Uninduced and fully induced L. lactis KF147 harboring pNZ6200 were not transformable after shortened, standard (2 h) and prolonged induction with nisin. Moderately induced L. lactis KF147 harboring pNZ6200 could be transformed after prolonged nisin induction, however, prolonged induction did not lead to increased transformation rates as compared with the standard incubation time.

    23. Pipet 100 µl of each sample into a semi-micro cuvette containing 900 µl GM17 and measure the OD600 of the sample or perform a total cell count.
      Note: Take 1 ml of GM17 as a new blank.
    24. Add the remaining 500 µl in the 5 ml recovery medium in a 15 ml tube and incubate for another 2 h at 30 °C in a water bath non-shaking.
      Note: When transforming a plasmid or PCR product that contains a resistance gene against bacteriostatic antibiotics, this recovery step is not necessary.
    25. Add 250 µl of the culture in a semi-micro cuvette containing 750 µl GM17 to measure OD600 or perform a total cell count.
    26. Pellet the rest of the culture in the 15 ml CELLSTAR® Polypropylene Tube by centrifugation at 4,000 x g for 10 min.
    27. Discard supernatant, leave 100 µl medium onto the pellet, resuspend and plate all cells on GM17A (M17 agar supplemented with 2% glucose) with appropriate antibiotics.
    28. Keep the plates in the incubation stove at 30 °C for 2 to 3 days.
    29. Calculate the transformation rate as (transformants/total cell number after induction/µg plasmid DNA).

    Data analysis

    1. Sequence similarity of competence proteins from the strain of interest to query sequences BlastP analysis should be performed in order to assess the completeness of the competence system in the strain of interest by comparing its competence protein sequences to the query sequences from either L. lactis subsp. lactis KF147 or L. lactis subsp. cremoris KW2 (Appendix 1; 2; 3). As previously described, a virtually full-length alignment (> 90%) of the subject sequence to the query sequence is considered indicative of gene presence (Mulder et al., 2017). Commonly, e-values of 10-5 indicate significant alignments. If desired, genetic events leading to decay of competence genes can be further analyzed by bioinformatics software such as Clone Manager suite.
    2. Calculation of transformation rates
      At least three replicates should be included in each experiment in order to calculate the average transformation rate of a nisin-induced L. lactis strain. Transformation rates can be shown either in tables or (in case of multiple time points) in a graph as depicted in Figure 1 by using Graphpad Prism or any other software.

    Notes

    1. Preparation of electrocompetent cells of L. lactis was based on Wells et al., 1993.
    2. If a strain displays poor growth in the glycine-containing medium (i.e., an overnight culture does not reach an OD600 of 0.7), the strain requires adaptation to GSGM17 medium by diluting this medium with regular GM17 (M17 supplemented with 2% glucose) 1:4, followed by serial subculturing in media with increasing amounts of glycine, to eventually reach the 0.4 M glycine concentration in GSGM17. If cells fail to adapt to the presence of 0.4 M glycine in GSGM17, then use the highest concentration of glycine possible.
    3. The L. lactis strain of interest should harbor a complete set of competence genes to allow competence induction upon comX overexpression. Completeness of the com gene set can be assessed by BlastP analysis using reference sequences for either subsp. lactis (Appendix 1) or subsp. cremoris as query (Appendix 2).
    4. The analogous alternative plasmid for pNZ9531, pNZ9530, can likely also be used to obtain a nisRK+ strain. In fact, it is expected that a tighter control of nisRK expression can be achieved when using pNZ9530 (Kleerebezem et al., 1997a).
    5. For extraction of pNZ9530 or pNZ9531 (low and medium copy plasmids, respectively) from an L. lactis strain: inoculate this strain in 25 ml GM17 supplemented with 10 µg/ml erythromycin without agitation. The next day, prepare 4 bottles of 200 ml GM17 supplemented with 10 µg/ml erythromycin and transfer 5 ml overnight culture to each bottle. Culture cells to an OD600 of 0.5-1 and pellet cells by centrifugation at 5,000 x g for 15 min. Proceed with Step 3 from the Plasmid DNA Isolation procedure.
    6. Presence of a chromosomal copy of nisRK in the L. lactis strain of interest can be examined (use Appendix 3 for query sequences of NisR and NisK for a BlastP analysis). If the strain of interest harbors nisRK, pNZ9530/pNZ9531 and subsequent transformation with one of these plasmids is not necessary.
    7. However, if the strain harbors nisRK which is also a nisin producer, this might result in constitutive induction of comX which may not lead to the favorable conditions for competence induction. In this case, an alternative could be transforming cells with pGIBLD001 (constitutive expression of comX under P32 control [David et al., 2017]).
    8. If for whatever reason GCDM cannot be used as culturing medium for L. lactis competence, M17 can be used, however, transformation rates are reduced by at least 10 fold for L. lactis KF147 harboring pNZ6200 (data not shown). If competence induction needs to be performed in medium that does not contain fluorescent components such as riboflavin, we recommend using CDMPC (chemically defined medium prolonged cultivation [Goel et al., 2012]) though transformation rates are reduced (1 x 10-7 transformants/total cell number after induction/µg plasmid DNA) for L. lactis KF147 harboring pNZ6200 in this medium.
    9. Induction of competence in L. lactis can also be performed in a high-throughput setup. However, we recommend inducing the culture in the 12 ml Cell Culture Tubes first, collect a 600 µl sample of the induced culture to add 1 µg DNA and seed 3 times 200 µl each time into a 96 wells plate. After 3 h of induction, 5 µl can be used for spot plating on GM17 plates with appropriate antibiotics by using a multichannel pipet. Centrifuge the rest of the culture in a plate centrifuge for 10 min at 3,700 x g and discard 150 µl supernatant. Resuspend the pelleted cells in the leftover medium and spot with a multichannel pipet 5 µl on a GM17 plate with appropriate antibiotics. For L. lactis KF147 harboring pNZ6200, we were able to obtain similar transformation rates (1.5 x 10-6 ± 4.0 x 10-7 transformants/total cell number after induction/µg plasmid DNA) as reported previously (Mulder et al., 2017). Integration of linear DNA fragments harboring an antibiotic resistance marker flanked by homologous regions can also be performed by using this protocol. For L. lactis KF147 harboring pNZ6200 natural transformation rates are typically similar to those obtained with pNZ6202 (Mulder et al., 2017).

    Recipes

    1. GSGM17 (1 L)
      M17 medium
      2% glucose
      0.5 M sucrose (= 170 g)
      0.4 M glycine (= 30 g)
      Autoclave at 121 °C for 15 min
    2. Washing solution 1 (500 ml)
      0.5 M sucrose (= 85 g)
      10% v/v glycerol
      RO (reversed osmosis) water
      Autoclave at 121 °C for 15 min
    3. Washing solution 2 (500 ml)
      0.5 M sucrose (= 85 g)
      0.05 M EDTA
      10% v/v glycerol
      RO (reversed osmosis) water
      Autoclave at 121 °C for 15 min
    4. Recovery medium
      GM17
      Note: GM17 is the same as M17 broth but then supplemented with 0.5 % glucose (w/v).
      20 mM MgCl2
      2 mM CaCl2
    5. Chemically Defined Medium (CDM) (500 ml)
      Note: The recipe for CDM for L. lactis is based on Otto et al., 1983; Poolman and Konings, 1988.

      Add up to 500 ml with RO (reversed osmosis) water
      Adjust the pH to 6.8
      Filter sterilize 500 ml in a sterile 500 ml bottle
    6. GCDM (chemically defined medium supplemented with glucose)
      Add sterilized glucose (20% solution, Tritium) to a final concentration of 2% to CDM prior to culturing L. lactis cells to obtain GCDM
    7. Stock solutions for CDM
      Note: *Solutions that need to be prepared on the day of use.
      1. Nucleotide solution*

        Make fresh and then use 5 ml
      2. MnCl2·4H2O solution*

      3. Amino acid solution

        Dissolve at a 6.5 pH (allow 1 h) at room temperature
        Aliquot into 50 ml CELLSTAR® Polypropylene tubes and store at -20 °C
      4. Metal solution

        Aliquot into 6 ml tubes and store at -20 °C
      5. Iron solution

        Aliquot into 6 ml tubes and store at -20 °C
      6. Vitamin solution

        Increase the pH until all components are dissolved. Afterwards, adjust the pH to 7.0
        Aliquot into 6 ml tubes and store at -20 °C

    Acknowledgments

    We acknowledge Sabri Cebeci and Koen Giesbers of NIZO for technical assistance. This work was carried out within the BE-Basic R&D Program, which was granted an FES subsidy from the Dutch Ministry of Economic Affairs. This protocol was adapted from Mulder et al. (2017).The authors have no conflicts of interest to declare.

    References

    1. Blokesch, M. (2016). Natural competence for transformation. Curr Biol 26(21): R1126-R1130.
    2. Blomqvist, T., Steinmoen, H. and Havarstein, L. S. (2006). Natural genetic transformation: A novel tool for efficient genetic engineering of the dairy bacterium Streptococcus thermophilus. Appl Environ Microbiol 72(10): 6751-6756.
    3. David, B., Radziejwoski, A., Toussaint, F., Fontaine, L., Henry de Frahan, M., Patout, C., van Dillen, S., Boyaval, P., Horvath, P., Fremaux, C. and Hols, P. (2017). Natural DNA transformation is functional in Lactococcus lactis ssp. cremoris KW2. Appl Environ Microbiol.
    4. Fontaine, L., Dandoy, D., Boutry, C., Delplace, B., de Frahan, M. H., Fremaux, C., Horvath, P., Boyaval, P. and Hols, P. (2010). Development of a versatile procedure based on natural transformation for marker-free targeted genetic modification in Streptococcus thermophilus. Appl Environ Microbiol 76(23): 7870-7877.
    5. Fontaine, L., Wahl, A., Flechard, M., Mignolet, J. and Hols, P. (2015). Regulation of competence for natural transformation in streptococci. Infect Genet Evol 33: 343-360.
    6. Goel, A., Santos, F., Vos, W. M., Teusink, B. and Molenaar, D. (2012). Standardized assay medium to measure Lactococcus lactis enzyme activities while mimicking intracellular conditions. Appl Environ Microbiol 78(1): 134-143.
    7. Håvarstein, L. S., Coomaraswamy, G. and Morrison, D. A. (1995). An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae. Proc Natl Acad Sci U S A 92(24): 11140-11144.
    8. Horton, R. M., Cai, Z. L., Ho, S. N. and Pease, L. R. (1990). Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques 8(5): 528-535.
    9. Johnston, C., Martin, B., Fichant, G., Polard, P. and Claverys, J. P. (2014). Bacterial transformation: distribution, shared mechanisms and divergent control. Nat Rev Microbiol 12(3): 181-196.
    10. Kleerebezem, M., Beerthuyzen, M. M., Vaughan, E. E., de Vos, W. M. and Kuipers, O. P. (1997a). Controlled gene expression systems for lactic acid bacteria: transferable nisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp. Appl Environ Microbiol 63(11): 4581-4584.
    11. Kleerebezem, M., Quadri, L. E., Kuipers, O. P. and de Vos, W. M. (1997b). Quorum sensing by peptide pheromones and two-component signal-transduction systems in Gram-positive bacteria. Mol Microbiol 24(5): 895-904.
    12. Mierau, I. and Kleerebezem, M. (2005). 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl Microbiol Biotechnol 68(6): 705-717.
    13. Mulder, J., Wels, M., Kuipers, O. P., Kleerebezem, M. and Bron, P. A. (2017). Unleashing natural competence in Lactococcus lactis by induction of the competence regulator ComX. Appl Environ Microbiol.
    14. Otto, R., ten Brink, B., Veldkamp, H. and Konings, W. N. (1983). The relation between growth rate and electrochemical proton gradient of Streptococcus cremoris. FEMS Microbiol Lett 16(1): 69-74.
    15. Pestova, E. V., Havarstein, L. S. and Morrison, D. A. (1996). Regulation of competence for genetic transformation in Streptococcus pneumoniae by an auto-induced peptide pheromone and a two-component regulatory system. Mol Microbiol 21(4): 853-862.
    16. Poolman, B. and Konings, W. N. (1988). Relation of growth of Streptococcus lactis and Streptococcus cremoris to amino acid transport. J Bacteriol 170(2): 700-707.
    17. Seitz, P. and Blokesch, M. (2013). Cues and regulatory pathways involved in natural competence and transformation in pathogenic and environmental Gram-negative bacteria. FEMS Microbiol Rev 37(3): 336-363.
    18. Wells, J. M., Wilson, P. W. and Le Page, R. W. (1993). Improved cloning vectors and transformation procedure for Lactococcus lactis. J Appl Bacteriol 74(6): 629-636.

简介

在过度表达细菌能力的主要调节因子ComX后,天然能力可以在乳酸乳球菌亚种乳酸和 cremoris 中激活。 在本文中,我们展示了通过在 L中使用乳链菌肽诱导型启动子调节 comX 基因的表达来激活细菌能力的方法。 含有 nisRK 的染色体或质粒编码拷贝的lactis 菌株。 加入中等浓度的诱导剂乳链菌肽导致伴随的中等水平的ComX,其导致最佳转化率(1.0×10 2 sup / -6>转化子/总细胞数/ g质粒DNA)。 在此,提出了用于能力归纳的优化协议的详细描述。

【背景】自然能力是细菌通过专门的摄取机制获得外源DNA的过程,之后内化的DNA整合到其基因组中或作为质粒DNA维持。一些细菌在特定的环境触发因素如基因毒性应激或饥饿时进入能力状态(Seitz和Blokesch,2013; Blokesch,2016)。群体感应系统,如 comCDE 或 comRS ,控制着革兰氏阳性菌的自然能力的激活(Håvarstein et al。,1995; Pestova et al。,1996; Kleerebezem et al。,1997b; Fontaine et al。,2015)。更具体地说, comC 和 comS 编码信息素,而 comD 编码组氨酸激酶和 comE 和 comR 编码响应调节器(Håvarstein et al。,1995; Pestova et al。,1996; Fontaine et al。,2010; Fontaine et al。,2015)。在链球菌中,活化的调节因子驱动替代西格玛因子ComX的转录,后者反过来激活能力基因的转录,所述能力基因编码包含DNA摄取机制的蛋白质(Johnston 等人,,2014)。以前,使用过表达ComX的策略导致在 Streptococcus thermophilus (Blomqvist et al。,2006)和 L中成功引入外源DNA。乳酸(David et al。,2017; Mulder et al。,2017),尽管采用了不同的方法来实现其过表达。这些研究还表明, comX 的不同表达水平对转化率具有重要影响。例如,在我们的工作中,我们使用了 L.乳酸亚种 lactis KF147,一种允许 Ni sin- C 控制基因 E xpression系统(NICE)的菌株(Mierau和Kleerebezem,2005)并且染色体 nisRK (允许乳链菌肽诱导必不可少),但不产生乳链菌肽。在该菌株中,我们通过电转化在乳链菌肽诱导型 nisA 启动子的控制下引入了含有 comX 的载体pNZ6200,并且得到的菌株不能转化。完全 comX 诱导(2 ng / ml乳链菌肽),而在中等水平观察到最佳转化率(1.0 x 10 -6 转化子/总细胞数/ g质粒DNA)诱导(0.03ng / ml乳链菌肽)(Mulder et al。,2017)。此外,我们还证明了应用相同的策略适用于 L的 nisRK 导数。乳酸亚种 lactis IL1403,而 L的能力诱导。乳酸亚种 cremoris KW2需要先用pNZ9531转化菌株,表达 nisRK (Kleerebezem et al。,1997a),以使乳链菌肽诱导表达是comX 。为了评估其他 L。乳酸菌菌株可以变得天然有效,我们在此提供一种方法,其中包含前面描述的能力方案的所有细节(Mulder et al。,2017),可以帮助其他科学家释放能力。 L.他们感兴趣的乳酸菌菌株可能会阻止乳链菌肽诱导的ComX表达的实验问题。这种新开发的协议有望在 L的广泛小组中进行遗传接入。乳酸菌株具有能够通过整合具有侧翼为染色体同源DNA区域的抗生素抗性标记的线性DNA片段而快速一步构建基因置换突变体的效率。

关键字:自然感受态, 转化, 乳酸乳球菌, ComX 过表达, NICE系统

材料和试剂

  1. 移液器吸头
  2. 15毫升和50毫升CELLSTAR ®聚丙烯管(锥形)(Greiner Bio One International,CELLSTAR ®,产品目录号分别为:188271和227261)
  3. 塑料培养皿94 x 16带通风口轻型(Greiner Bio One International,目录号:633181)
  4. 12 ml细胞培养管(Greiner Bio One International,CELLSTAR ®,目录号:163160)
  5. 接种循环
  6. Eppendorf管®安全锁管1.5 ml(Eppendorf,目录号:0030120086)
  7. 电穿孔比色皿,2 mm间隙(Bio-Rad Laboratories,目录号:1652086)
  8. 半微量比色皿,1毫升(用于光密度测量)
  9. 细菌菌株: L.具有一整套能力基因的感兴趣的乳酸菌 L.含有pNZ6200(Mulder et al。,2017),pNZ6202(Mulder 等人,2017)和pNZ9531(Kleerebezem 等。 ,1997a)
  10. M17肉汤(M17)和M17琼脂(M17A)(氚,目录号分别为:M086.76.0200和M085.76.0200)
  11. 葡萄糖溶液(20%,氚,目录号:G209.65.0080)
  12. 蒸馏水(Thermo Fisher Scientific,Gibco TM ,目录号:15230089)
  13. 抗生素储备液:
    20 mg / ml氯霉素(Sigma-Aldrich,目录号:C0378-100G)
    20mg / ml红霉素(Fisher Scientific,目录号:BP920-25)
    12.5 mg / ml盐酸四环素(Sigma-Aldrich,目录号:T7660-25G)
  14. NisinA ® P超纯Nisin A(Handary,布鲁塞尔,比利时,在含有0.05冰醋酸的蒸馏水中制备2 mg / ml储备液)
  15. 或者:来自乳酸乳球菌 2.5%的乳链菌肽(Sigma-Aldrich,目录号:N5764)
  16. JETSTAR 2.0 Maxiprep试剂盒(GENPRICE,目录号:220 020)或PureLink TM HiPure Plasmid Maxiprep试剂盒(Thermo Fisher Scientific,Invitrogen TM ,目录号:K210007)
  17. Qubit TM dsDNA BR检测试剂盒(Thermo Fisher Scientific,Invitrogen TM ,目录号:Q32850)
  18. Phenol BioUltra,用于分子生物学,TE饱和,~73%(T)(Sigma-Aldrich,目录号:77607)
  19. 氯仿HPLC级,≥99.9%(Sigma-Aldrich,目录号:528730)
  20. 溴化乙锭(Sigma-Aldrich,目录号:E1510-10ML)
  21. 琼脂糖片(美国生物技术资源,目录号:G01PD-500)
  22. 冰醋酸(Scharlab,目录号:AC03522500)
  23. β-甘油磷酸盐(二钠盐)(Sigma-Aldrich,目录号:50020-500G)
  24. 磷酸氢二钾(K 2 HPO 4 )(默克,目录号:1.05104.1000)
  25. 磷酸二氢钾(KH 2 PO 4 )(默克,目录号:1.04873.1000)
  26. 醋酸钠(默克,目录号:1.06268.1000)
  27. (NH 4 ) 3 - 柠檬酸(Sigma-Aldrich,目录号:A1332-500G)
  28. 抗坏血酸(VWR,AnalaR NORMPAPUR ®,目录号:20150.231)
  29. 氯化锰(II)四水合物(MnCl 2 ·4H 2 O)(Sigma-Aldrich,目录号:M8054-100G)
  30. 腺嘌呤(Sigma-Aldrich,Fluka,目录号:01830)
  31. 鸟嘌呤(Sigma-Aldrich,目录号:G11950-100G)
  32. Uracil(Sigma-Aldrich,Fluka,目录号:94220)
  33. 黄嘌呤(Sigma-Aldrich,目录号:X7375-10G)
  34. 丙氨酸(Sigma-Aldrich,目录号:A7627-100G)
  35. 精氨酸(Sigma-Aldrich,目录号:A5006-500G)
  36. 天冬氨酸(Sigma-Aldrich,目录号:A9256-100G)
  37. 半胱氨酸-HCl(Sigma-Aldrich,目录号:C1276-250G)
  38. 谷氨酸(Sigma-Aldrich,目录号:G1251-500G)
  39. 甘氨酸(默克,目录号:1.04201.0250)
  40. 组氨酸(Sigma-Aldrich,目录号:H8000-100G)
  41. 亮氨酸(Sigma-Aldrich,目录号:L8000-100G)
  42. 赖氨酸(Sigma-Aldrich,目录号:L5626-100G)
  43. 蛋氨酸(Sigma-Aldrich,目录号:M9625-100G)
  44. 苯丙氨酸(Sigma-Aldrich,目录号:P2126-100G)
  45. 脯氨酸(Sigma-Aldrich,目录号:P0380-100G)
  46. 丝氨酸(Sigma-Aldrich,目录号:S4500-100G)
  47. 苏氨酸(Sigma-Aldrich,目录号:T8625-100G)
  48. 色氨酸(Sigma-Aldrich,目录号:T0254-100G)
  49. 缬氨酸(Sigma-Aldrich,目录号:V0500-100G)
  50. 氯化镁六水合物(MgCl 2 ·6H 2 O)(Sigma-Aldrich,目录号:M2670-100G)
  51. 氯化钙二水合物(CaCl 2 ·2H 2 O)(默克,目录号:1.02382.0500)
  52. 硫酸锌七水合物(ZnSO 4 ·7H 2 O)(Sigma-Aldrich,目录号:Z0251-100G)
  53. 硫酸钴(II)七水合物(CoSO 4 ·7H 2 O)(Sigma-Aldrich,目录号:C6768-100G)
  54. 硫酸铜(II)五水合物(CuSO 4 ·5H 2 O)(Scharlab,目录号:CO01010500)
  55. 钼酸铵四水合物((NH 4 ) 6 Mo 7 O 24 ·4H 2 O)(Sigma-Aldrich,Fluka,目录号:09878)
  56. 氯化铁(II)四水合物(FeCl 2 ·4H 2 O)(Sigma-Aldrich,目录号:44939-50G)
  57. 盐酸(HCl)(Sigma-Aldrich,目录号:30721-1L)
  58. 氯化铁(III)六水合物(FeCl 3 ·6H 2 O)(Sigma-Aldrich,目录号:F2877-100G)
  59. 对氨基苯甲酸(Sigma-Aldrich,目录号:A9879-100G)
  60. 肌苷(Sigma-Aldrich,目录号:I4125-25G)
  61. 乳清酸(Sigma-Aldrich,目录号:O2750-100G)
  62. 吡哆胺-HCl(Sigma-Aldrich,目录号:P9380-5G)
  63. 胸苷(Sigma-Aldrich,目录号:T9250-10G)
  64. D-生物素(Sigma-Aldrich,目录号:B4501-5G)
  65. 6,8-硫辛酸(Sigma-Aldrich,目录号:T5625-5G)
  66. 吡哆醇-HCl(Sigma-Aldrich,目录号:P9755-25G)
  67. 叶酸(Sigma-Aldrich,目录号:F7876-25G)
  68. 烟酸(Sigma-Aldrich,目录号:N4126-5G)
  69. Ca-(D +)泛酸盐(Sigma-Aldrich,目录号:P5155-100G)
  70. 核黄素(Sigma-Aldrich,目录号:R4500-25G)
  71. 硫胺-HCl(Sigma-Aldrich,目录号:T4625-100G)
  72. 维生素B12(Sigma-Aldrich,目录号:V2876-5G)
  73. 或者,PCR产物(用KOD热启动DNA聚合酶获得)
    注意:含有抗生素抗性标记,其侧翼为染色体片段,通过重叠PCR获得整合(Horton等,1990),如果优于质粒转化,例如,用于构建基因置换突变体。 < br />
  74. 可选:KOD热启动DNA聚合酶Master Mix(Merck,Novagen,目录号:71842-4)
  75. GSGM17(见食谱)
  76. 洗涤液1(见食谱)
  77. 洗涤液2(见食谱)
  78. 回收介质(见食谱)
  79. CDM(见食谱)
  80. GCDM(化学成分确定的培养基补充葡萄糖)
  81. 清洁发展机制的储备解决方案(见食谱)
    1. 核苷酸溶液
    2. MnCl 2 ·4H 2 O溶液
    3. 氨基酸溶液
    4. 金属解决方案
    5. 铁解决方案
    6. 维生素溶液

设备

  1. 普通移液器
  2. 200毫升的瓶子
  3. 水浴(30°C和55°C)
  4. 孵化炉在30°C
  5. 涡旋
  6. 高压灭菌器
  7. 微量离心机(Eppendorf ®冷冻微量离心机)(Eppendorf,型号:5417R)
  8. 离心15和50毫升管(Heraeus Megafuge 1.0R)
  9. Qubit ® 2.0荧光计(Thermo Fisher Scientific,Invitrogen TM ,模式:Qubit ® 2.0)
  10. Electroporator(Bio-Rad Laboratories,型号:GenePulserXcell TM )
  11. 分光光度计(Genesys 10紫外分光光度计)
  12. Nanodrop ND1000分光光度计(NanoDrop Technologies)(Thermo Fisher Scientific,型号:NanoDrop TM 1000

程序

  1. 质粒DNA分离
    1. 文化 L.乳酸含有pNZ6200,pNZ6202或pNZ9531,在含有适当抗生素(表1)的200毫升瓶中,在含有适当抗生素(表1)的200毫升M17中加入1%葡萄糖,不振摇过夜。

      表1.本协议中使用的质粒特征


    2. 通过在室温下以5,000 x g 离心15分钟沉淀细胞。
    3. 弃去培养上清液,将颗粒在-20°C冷冻至少30分钟。
    4. 将沉淀重悬于含有40mg溶菌酶的10ml重悬浮缓冲液(来自Maxiprep试剂盒的制造商溶液)中。
    5. 在55°C孵育1.5小时,不要摇晃。
    6. 根据制造商的方案,从JETSTAR 2.0 Maxiprep Kit或PureLink TM HiPure Plasmid Maxiprep Kit继续进行质粒分离。
    7. 在制造商的方案中加入一些额外步骤:将裂解液离心沉淀至细胞碎片后,对上清液进行苯酚 - 氯仿提取(苯酚:氯仿:上清液,体积比为1:1:1,混合物)通过倒置管)并在5,000 xg 下离心30分钟。将水相(上层)转移到新管中,并进行氯仿抽提(水相:氯仿1:1体积),随后在5,000 x g 下离心15分钟。从制造商的试剂盒中加载纯化的级分,以便结合DNA并继续执行手册中指示的步骤。
    8. 通过Qubit确定质粒DNA的DNA浓度(每次转化需要1μgDNA,其中质粒溶液的浓度基于Qubit测量)。
    9. 在1%琼脂糖凝胶上检查分离的质粒DNA的质量。

  2. pNZ6200和pNZ9531电转化为电感受态 L.乳酸细胞(图1)。


      图1.评估能力潜力(A)和获得 L的示意图。含有pNZ6200和pNZ9531的lactis 菌株,以及随后通过乳链菌肽诱导诱导能力(B)。有关我们建议修改此标准流程方案的特定情况,请参阅程序部分中的注释。 br />
    1. 准备1L电子能力生长培养基GSGM17,500ml洗涤溶液1和500ml洗涤溶液2(参见配方)。
    2. 文化 L.乳酸最初在10 ml GM17中在12 ml细胞培养管中过夜,在30°C下无需摇动即可孵育。
    3. 第二天,将5 ml培养物接种到50 ml GSGM17中,在30°C温育细胞过夜,不要摇晃。
    4. 如果过夜培养物达到OD 600 ≥0.7,则在GSGM17中以1:8稀释过夜培养物,并使培养物生长至OD 600 0.2-0.3。 >
    5. 通过在4℃下以6,000 x g 离心20分钟沉淀细胞。
    6. 用1体积冰冷的洗涤溶液洗涤沉淀1.
    7. 通过在4℃下以5,000 x g 离心20分钟来沉淀细胞。
    8. 用0.5体积冰冷的洗涤溶液2洗涤沉淀,并在冰上孵育15分钟。
    9. 通过在4℃下以5,000 x g 离心20分钟来沉淀细胞。
    10. 用0.25体积的冰冷洗涤溶液1洗涤沉淀,并通过在4℃下以5,000 x g 离心20分钟沉淀细胞。
    11. 将细胞重悬于0.01体积的冰冷洗涤溶液1中,每50μl等分细胞。
    12. 立即使用新鲜的电感受态细胞进行转化,获得所需的质粒。
      选项:存储细胞以供以后在-80°C下使用。
    13. 将电子比色皿置于冰上,制备含有补充有1%葡萄糖,200 mM MgCl 2 和20 mM CaCl 2 的M17的回收培养基,并将其保存在冰上。 br />
    14. 将1μg质粒加入到电感受态细胞中,并将带有DNA的细胞悬液转移到电子比色杯中。
      注意:在向电感受态细胞中加入质粒DNA后,不要上下移液。轻轻地轻弹管子,混合细胞和质粒DNA。
    15. 电穿孔;电穿孔装置的设置:电压= 2,000 V,容量=25μF,电阻=200Ω。
      1. 用纸巾擦干电子比色杯,轻轻地将电子比色杯轻轻敲打到工作台上5次,将细胞滑落到电子比色杯的底部。
      2. 将电子比色杯放入电穿孔仪的比色皿支架中并施加电脉冲。
    16. 尽快将1毫升冰冷的回收培养基加入电子比色杯中,然后将电子比色杯再次放在冰上。
    17. 将电池中的细胞吸移到1.5 ml Eppendorf管中,在30°C水浴中孵育样品1小时,不要摇晃。
    18. 在含有适当抗生素的选择平板上的回收培养基中移取10 0 至10 -6 的系列稀释液。
    19. 在30°C非振荡条件下孵育平板2-3天。
    20. 在GM17琼脂平板上清洁条纹阳性菌落,补充适当的抗生素。
      注意:如果pNZ9531和pNZ6200的共转化不成功,首先用pNZ9531转化细胞。在GM17中选择阳性菌落和培养菌落,补充有10μg/ ml红霉素。随后,重复电泳能力细胞的制备,并通过电穿孔引入pNZ6200。
    21. 第二天,从GM17清洁条纹平板培养单菌落,补充适当的抗生素,在30°C,非摇动条件下孵育过夜。
    22. 准备25%甘油原液并储存在-80°C。

  3. L中自然能力的诱导。球菌
    1. 准备GCDM(参见食谱)并用适当的抗生素制备GM17琼脂平板。
    2. 文化 L.乳酸分别在含有适当抗生素的10 ml GCDM中含有pNZ6200和pNZ9531(表1)。
    3. 准备一个含有50毫升GCDM的50毫升CELLSTAR ®聚丙烯管。
    4. 将细胞和含有50 ml GCDM的50 ml CELLSTAR ®聚丙烯管在30°C温育培养炉中过夜孵育过夜。
    5. 第二天,将氯霉素和红霉素加入含50 ml GCDM的50 ml CELLSTAR ®聚丙烯管中。
    6. 通过在OD 600 下测量光密度,在半微量比色杯(=空白样品)中加入1 ml GCDM。
    7. 加入750μl L.乳酸将pNZ6200过夜培养物置于含有GCDM的50 ml CELLSTAR ®聚丙烯管中。
    8. 通过倒置封闭的管混合细胞培养物。
    9. 将1ml培养物吸移到半微量比色皿中并在30℃水浴中不振摇培养细胞直至培养物达到OD <600> 0.3(约3-4小时后)。
    10. 测量培养物的初始OD 600 。
      注意:t = 0时文化的OD 600 应该在0.03左右。如果OD 600 低于0.03;增加更多文化。
    11. 在非摇动条件下培养细胞直至OD 600 为0.3(对应于约2-3×10 8 细胞)。
    12. 准备计算和恢复培养基(参见食谱,每次转化5毫升回收培养基)。
    13. 将OD 600 0.3的10ml细胞培养物转移到12ml无菌管中。
    14. 重复此步骤以获得总共3个等分培养物。
      注意:这些培养物将用于后续步骤,包括用0 ng / ml(阴性对照),0.03 ng / ml(最佳)和2 ng / ml(完全诱导)乳链菌肽诱导comX表达。
    15. 随后,使用NisinA ® P超纯乳链菌肽A或来自乳酸乳球菌 2.5的乳链菌肽制备 乳链菌肽稀释液%(Sigma-Aldrich,2.5%乳链菌肽)。
    16. 使用含有0.05%冰醋酸的蒸馏水在玻璃管中制备乳链菌肽稀释液,以提高乳链菌肽溶液的稳定性。
      注意:当使用2.5%乳酸乳球菌(Sigma-Aldrich)的乳链菌肽时,请确保校正稀释因子(40x),因为只有2.5%的干物质是乳链菌肽。
    17. 用乳链菌肽诱导10ml培养物。为获得最佳转化率,用乳链菌肽诱导细胞至终浓度为0.03 ng / ml乳链菌肽。
      注意:
      1. 可选:作为对照,包括未诱导的样品和完全诱导的样品(2 ng / ml)。
      2. 升。含有2 ng / ml乳链菌肽诱导的pNZ6200的乳酸菌KF147将在诱导1 h后停止生长,但其他菌株可能在此浓度下继续生长。
    18. 加入乳链菌肽后,立即将管倒置3次。
    19. 将600μl诱导培养物放入Eppendorf管中。
    20. 加入1μgpNZ6202。
      注意:其他兼容的质粒很可能也可用于评估转化效率。
    21. 通过倒置管进行混合。
    22. 在30°C下在水浴中孵育2小时,不要摇晃。
      注意:与乳链菌肽进行更长时间的孵育不会导致转化率增加(见图2)。


      图2.在 L中延长乳链菌肽诱导后的转化率(转化体/诱导后的总细胞数/μg质粒DNA)。含有pNZ6200的乳酸菌KF147 在未诱导的(实心圆圈),中度诱导的(实心方块)和完全诱导的(实心三角形)培养物中检查转化。未诱导和完全诱导 L.含有pNZ6200的乳酸 KF147在缩短,标准(2小时)和用乳链菌肽延长诱导后不能转化。中度诱导 L.含有pNZ6200的乳酸菌KF147可在长时间的乳链菌肽诱导后转化,但与标准培养时间相比,延长诱导不会导致转化率增加。

    23. 将100μl每种样品吸取到含有900μlGM17的半微量比色杯中,测量样品的OD 600 或进行总细胞计数。
      注意:取1毫升GM17作为新的空白。
    24. 将剩余的500μl加入5 ml回收培养基中的15 ml试管中,在30°C水浴中再摇动2小时,不要摇晃。
      注意:转化含有抑菌抗生素抗性基因的质粒或PCR产物时,不需要进行此恢复步骤。
    25. 在含有750μlGM17的半微量比色皿中加入250μl培养物,测量OD 600 或进行总细胞计数。
    26. 将剩余的培养物在15 ml CELLSTAR ®聚丙烯管中沉淀,在4,000 x g 下离心10分钟。
    27. 弃去上清液,将100μl培养基留在沉淀上,重悬浮并用适当的抗生素将所有细胞培养在GM17A(M17琼脂补充2%葡萄糖)上。
    28. 将培养皿中的培养板保持在30°C下2至3天。
    29. 将转化率计算为(转化子/诱导后的总细胞数/μg质粒DNA)。

数据分析

  1. 来自目标菌株的能力蛋白质与查询序列的序列相似性应该进行BlastP分析以通过将其能力蛋白质序列与来自 L的查询序列进行比较来评估目标菌株中能力系统的完整性。乳酸亚种 lactis KF147或 L.乳酸亚种 cremoris KW2(附录 1 ; 2 ; 3 )。如前所述,主题序列与查询序列的几乎全长比对(> 90%)被认为是基因存在的指示(Mulder 等人,2017)。通常,10 -5 的e值表示显着的比对。如果需要,可以通过生物信息学软件(如克隆管理器套件)进一步分析导致能力基因衰退的遗传事件。
  2. 转换率的计算
    每个实验中应包括至少三次重复,以计算乳链菌肽诱导的 L的平均转化率。乳酸菌菌株。如图1所示,通过使用Graphpad Prism或任何其他软件,可以在表格中或(在多个时间点的情况下)显示转换率。

笔记

  1. L的电感受态细胞的制备。 lactis 基于Wells et al。,1993。
  2. 如果菌株在含甘氨酸的培养基中显示出不良生长(即,则过夜培养物未达到OD 600 0.7),该菌株需要通过以下方法适应GSGM17培养基:用常规GM17(补充有2%葡萄糖的M17)1:4稀释该培养基,然后在含有增加量甘氨酸的培养基中连续传代培养,最终达到GSGM17中的0.4M甘氨酸浓度。如果细胞不能适应GSGM17中0.4 M甘氨酸的存在,那么可能使用最高浓度的甘氨酸。
  3. L.感兴趣的乳酸菌株应具有一套完整的能力基因,以便在 comX 过表达时诱导能力。 com 基因集的完整性可以通过使用任一亚种的参考序列的BlastP分析来评估。 lactis (附录1 )或亚种。 cremoris 作为查询(附录2 )。
  4. pNZ9531的类似替代质粒pNZ9530也可能用于获得 nisRK + 菌株。事实上,当使用pNZ9530时,预计可以实现对 nisRK 表达的更严格控制(Kleerebezem et al。,1997a)。
  5. 用于从 L中提取pNZ9530或pNZ9531(分别为低和中拷贝质粒)。 lactis 菌株:将该菌株接种于25ml补充有10μg/ ml红霉素的GM17中,无需搅拌。第二天,准备4瓶200ml GM17,补充10μg/ ml红霉素,并将5ml过夜培养物转移到每个瓶中。将细胞培养至OD1 <600> 0.5-1,并通过在5,000×g离心15分钟沉淀细胞。从质粒DNA分离程序继续步骤3。
  6. 在 L中存在 nisRK 的染色体拷贝。可以检查感兴趣的lactis 菌株(使用附录3 用于BlastP分析的NisR和NisK查询序列)。如果感兴趣的菌株含有 nisRK ,那么pNZ9530 / pNZ9531不需要用这些质粒中的一种进行转化。
  7. 然而,如果菌株还含有也是乳链菌肽生产者的 nisRK ,这可能导致 comX 的组成型诱导,这可能不会导致能力诱导的有利条件。在这种情况下,替代方案可以是用pGIBLD001转化细胞( P 32 对照下的 comX 的组成型表达[David et al。 ,2017])。
  8. 如果由于某种原因GCDM不能用作 L的培养基。乳酸能力,可以使用M17,然而, L的转化率降低至少10倍。含有pNZ6200的乳酸 KF147(数据未显示)。如果需要在不含核黄素等荧光成分的培养基中进行能力诱导,我们建议使用CDMPC(化学成分确定培养基延长培养[Goel 等,2012])尽管转化率降低(1×10 -7 转化体/诱导后的总细胞数/μg质粒DNA) L.乳酸 KF147在该培养基中含有pNZ6200。
  9. L的能力归纳。 lactis 也可以在高通量设置中进行。然而,我们建议首先在12 ml细胞培养管中诱导培养物,收集600μl诱导培养物样品,每次加入1μgDNA和3次200μl种子到96孔板中。诱导3小时后,通过使用多通道移液管,5μl可用于在具有适当抗生素的GM17板上进行点镀。将离心培养物中的其余培养物在板式离心机中以3,700 x g 离心10分钟,弃去150μl上清液。将沉淀的细胞重新悬浮在剩余的培养基中,用含有适当抗生素的GM17平板上的5μl多通道移液管进行检测。对于 L.含有pNZ6200的乳酸菌KF147,我们能够获得相似的转化率(1.5×10 -6 ±4.0×10 -7 转化体/诱导后的总细胞数如先前所报道的(μg质粒DNA)(Mulder 等人,,2017)。具有侧翼为同源区域的抗生素抗性标记的线性DNA片段的整合也可以通过使用该方案进行。对于 L.乳酸 KF147含有pNZ6200的自然转化率通常与pNZ6202相似(Mulder et al。,2017)。

食谱

  1. GSGM17(1 L)
    M17中等
    2%葡萄糖
    0.5M蔗糖(= 170g)
    0.4M甘氨酸(= 30g)
    在121°C高压灭菌15分钟
  2. 洗涤液1(500毫升)
    0.5M蔗糖(= 85g)
    10%v / v甘油
    RO(反渗透)水
    在121°C高压灭菌15分钟
  3. 洗涤液2(500毫升)
    0.5M蔗糖(= 85g)
    0.05 M EDTA
    10%v / v甘油
    RO(反渗透)水
    在121°C高压灭菌15分钟
  4. 恢复介质
    GM17
    注意:GM17与M17肉汤相同,但补充0.5%葡萄糖(w / v)
    20mM MgCl 2
    2 mM CaCl 2
  5. 化学定义培养基(CDM)(500 ml)
    注意:乳酸乳球菌的CDM配方基于Otto等,1983; Poolman和Konings,1988年

    用RO(反渗透)水加入500毫升
    将pH调节至6.8
    过滤500毫升无菌500毫升瓶消毒
  6. GCDM(化学成分确定的培养基补充葡萄糖)
    在培养乳酸乳球菌细胞之前,将灭菌的葡萄糖(20%溶液,氚)加至终浓度为2%至CDM,以获得GCDM
  7. CDM的股票解决方案
    注意:*需要在使用当天准备的解决方案
    1. 核苷酸溶液*

      新鲜,然后使用5毫升
    2. MnCl 2 ·4H 2 O溶液*

    3. 氨基酸溶液

      在室温下溶解于6.5 pH(允许1小时)
      将其分装到50 ml CELLSTAR ®聚丙烯管中,并储存在-20°C
    4. 金属解决方案

      分装到6毫升管中,储存在-20°C
    5. 铁解决方案

      分装到6毫升管中,储存在-20°C
    6. 维生素溶液

      增加pH值直至所有组分溶解。然后,将pH调节至7.0
      将其分装到6ml管中并在-20℃下储存

致谢

我们感谢NIZO的Sabri Cebeci和Koen Giesbers提供技术支持。这项工作是在BE-Basic R&amp; D计划内进行的,该计划获得了荷兰经济部的FES补贴。该协议改编自Mulder et al。(2017)。作者没有任何利益冲突申报。

参考

  1. Blokesch,M。(2016)。 转型的自然能力。 Curr Biol 26(21 ):R1126-R1130。
  2. Blomqvist,T.,Steinmoen,H。和Havarstein,L。S.(2006)。 自然遗传转化:一种用于乳酸菌嗜热链球菌的高效基因工程的新工具。 Appl Environ Microbiol 72(10):6751-6756。
  3. David,B.,Radziejwoski,A.,Toussaint,F.,Fontaine,L.,Henry de Frahan,M.,Patout,C.,van Dillen,S.,Boyaval,P.,Horvath,P.,Fremaux, C.和Hols,P。(2017)。 天然DNA转化在 Lactococcus lactis ssp中有功能。 cremoris KW2。 Appl Environ Microbiol 。
  4. Fontaine,L.,Dandoy,D.,Boutry,C.,Delplace,B.,de Frahan,M.H.,Fremaux,C.,Horvath,P.,Boyaval,P。and Hols,P。(2010)。 开发基于自然转化的多功能程序,用于链球菌的无标记靶向基因修饰嗜热菌。 Appl Environ Microbiol 76(23):7870-7877。
  5. Fontaine,L.,Wahl,A.,Flechard,M.,Mignolet,J。和Hols,P。(2015)。 调节链球菌自然转化的能力。 感染Genet Evol 33:343-360。
  6. Goel,A.,Santos,F.,Vos,W。M.,Teusink,B。和Molenaar,D。(2012)。 用于测量乳酸乳球菌酶活性的标准化分析培养基,同时模拟细胞内条件。 Appl Environ Microbiol 78(1):134-143。
  7. Håvarstein,L。S.,Coomaraswamy,G。和Morrison,D。A.(1995)。 未经修饰的十七肽信息素可诱导肺炎链球菌的遗传转化能力。; Proc Natl Acad Sci USA 92(24):11140-11144。
  8. Horton,R.M.,Cai,Z.L.,Ho,S.N。和Pease,L.R。(1990)。 通过重叠延伸进行基因剪接:使用聚合酶链式反应定制基因。 ; Biotechniques 8(5):528-535。
  9. Johnston,C.,Martin,B.,Fichant,G.,Polard,P。和Claverys,J.P。(2014)。 细菌转化:分布,共享机制和发散控制。 Nat Rev Microbiol 12(3):181-196。
  10. Kleerebezem,M.,Beerthuyzen,M。M.,Vaughan,E。E.,de Vos,W。M. and Kuipers,O。P.(1997a)。 乳酸菌的受控基因表达系统: Lactococcus的可转移的乳链菌肽诱导表达盒, Leuconostoc 和 Lactobacillus spp。 Appl Environ Microbiol 63(11):4581-4584。
  11. Kleerebezem,M.,Quadri,L.E.,Kuipers,O。P. and de Vos,W。M.(1997b)。 通过肽信息素和革兰氏阳性细菌中的双组分信号转导系统进行群体感应。 Mol Microbiol 24(5):895-904。
  12. Mierau,I。和Kleerebezem,M。(2005)。 乳酸乳球菌中乳酸链球菌素控制的基因表达系统(NICE)10年。 Appl Microbiol Biotechnol 68(6):705-717。
  13. Mulder,J.,Wels,M.,Kuipers,O.P.,Kleerebezem,M。and Bron,P。A.(2017)。 通过诱导能力调节剂ComX释放乳酸乳球菌的自然能力。 Appl Environ Microbiol 。
  14. Otto,R.,ten Brink,B.,Veldkamp,H。和Konings,W。N.(1983)。 Streptococcus cremoris 的生长速率与电化学质子梯度之间的关系。; FEMS Microbiol Lett 16(1):69-74。
  15. Pestova,E。V.,Havarstein,L。S.和Morrison,D。A.(1996)。 通过自身诱导调节肺炎链球菌遗传转化的能力肽信息素和双组分调节系统。 Mol Microbiol 21(4):853-862。
  16. Poolman,B。和Konings,W。N.(1988)。 乳酸链球菌和 Streptococcus cremoris的生长关系到氨基酸转运。 J Bacteriol 170(2):700-707。
  17. Seitz,P。和Blokesch,M。(2013)。 与致病性和环境革兰氏阴性菌的自然能力和转化相关的线索和调节途径。 FEMS Microbiol Rev 37(3):336-363。
  18. Wells,J.M.,Wilson,P.W。和Le Page,R.W。(1993)。 改进的乳酸乳球菌的克隆载体和转化程序。 J Appl Bacteriol 74(6):629-636。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mulder, J., Wels, M., Kuipers, O. P., Kleerebezem, M. and Bron, P. A. (2018). Induction of Natural Competence in Genetically-modified Lactococcus lactis. Bio-protocol 8(13): e2922. DOI: 10.21769/BioProtoc.2922.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。