搜索

Construction and Screening of a Transposon Insertion Library of Yersinia enterocolitica (YeO3-R1)
小肠结肠炎耶尔森菌 (YeO3-R1)转座子插入库的建立和筛选   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

The Mu-transposon system is one of the best characterized transposition systems. Under minimal in vitro set-up, Mu transposition requires only a simple reaction buffer, MuA transposase protein, mini-Mu transposon DNA (donor) and target DNA. The reaction proceeds via initial assembly of the transposition complex that directs transposon integration into target DNA with high efficiency and relatively low target site selectivity. These characteristics make the Mu in vitro transposition technology ideal for the generation of comprehensive mutant DNA libraries usable in a variety of molecular biology applications. This technology has successfully been used for DNA sequencing, functional analyses of plasmid DNA and virus genomes, protein engineering for structure/function and protein-protein interaction studies and generation of gene targeting constructions. When electroporated, the in vitro–assembled Mu transposition complexes can also be used for efficient gene delivery in bacteria, yeasts and mammalian cells. Using this protocol we have identified several mutants where Cat-Mu insertion has interrupted genes involved in lipopolysaccharide (LPS) biosynthesis (Pinta et al., 2012).

Keywords: Yersinia enterocolitica(小肠结肠炎耶尔森菌), Mu transposase(Mu转座), Transposon insertion library(转座子插入库), Lipopolysaccharide(脂多糖), Bacteriophage(噬菌体)

Materials and Reagents

  1. Yersinia enterocolitica strain YeO3-R1 (al-Hendy et al., 1992)
  2. Cat-Mu transposon (Haapa et al., 1999) or equivalent like Entranceposon Cam-R3 (Thermo Fisher Scientific, Finnzymes,  catalog number: F-778 )
  3. MuA transposase 1,100 ng/μl and MuA storage buffer (Thermo Fisher Scientific, Finnzymes, catalog number: F-750C )
  4. 99.5% glycerol BDH (catalog number:  24388.320 )
  5. 10% Triton X-100 (w/v) (F. Hoffmann-La Roche, catalog number:  11332481001 )
  6. Ultrapure 0.5 M EDTA (pH 8.0) (Life Technologies, Invitrogen™, catalog number:  15575 )
  7. DTT (Sigma-Aldrich, catalog number:  D0632 )
  8. Centricon YM-100 (100-kDa cutt off) (EMD Millipore, catalog number:  UFC210024PL )
  9. Polyethylene glycol (PEG) 6000 (Merck KGaA, catalog number:  8.07491 )
  10. NuSieve 3:1 Agarose (25 g) (Lonza, catalog number: 50091 )
  11. Albumin from bovine serum, BSA (Sigma-Aldrich, catalog number:  A7906 )
  12. Heparin (Sigma-Aldrich, catalog number:  H3393 )
  13. Ficoll PM 400 (Sigma-Aldrich, catalog number:  F4375 )
  14. Bacto-agar, Bacto-tryptone, and Bacto-yeast extract (BD DifcoTM)
  15. Chloramphenicol (Sigma-Aldrich, catalog number:  C0378 )
  16. Tryptic Soya Broth (Oxoid Limited, catalog number:  CM0129 )
  17. Enterocoliticin produced by Y. enterocolitica serotype O: 7, 8 strain 29930 (Strauch et al., 2001)
  18. MuA storage buffer
  19. Sodium acetate (NaAc)
  20. DNA ladder (New England Biolabs)
  21. Phosphate-buffered saline (PBS) (pH 7.4)
  22. TGD buffer (see Recipes)
  23. 5x complex buffer (see Recipes)
  24. 1 M DTT (see Recipes)
  25. 20% PEG6000 (w/v) – 2.5 M NaCl (5 ml) (see Recipes)
  26. SOB medium (1 L) without magnesium (see Recipes)
  27. 2 M Mg++ stock (see Recipes)
  28. 2 M glucose (100 ml) (see Recipes)
  29. SOC medium (100 ml) (see Recipes)
  30. Luria broth (LB) medium (1 L) (see Recipes)
  31. LB agar (LA) plates (1 L) (see Recipes)
  32. Chloramphenicol (Clm) solution (10 ml) (see Recipes)
  33. Tryptic soya broth (see Recipes)
  34. Phosphate-buffered saline (PBS) (pH 7.4) (1 L) (see Recipes)
  35. TAE buffer (see Recipes)

Equipment

  1. Centrifuges (Sorvall, Heraeus Holding)
  2. Water bath (Grant)
  3. Incubator (Termaks)
  4. Shaker (New Brunswick Scientific)
  5. Biofotometer (Eppendorf)
  6. Agarose gel electrophoresis (Bio-Rad Laboratories)
  7. Gel documentation equipment (Bio-Rad Laboratories)
  8. Electroporator (Bio-Rad Laboratories, Genepulser II)
  9. 90 mm Petri dishes (Thermo Fisher Scientific, Sterilin®, catalog number: 101RT )
  10. 0.1-cm electroporation cuvettes (Bio-Rad Laboratories)

Procedure

  1. Transposition complex assembly (Lamberg et al., 2002)
    1. Prepare eight standard assembly reactions using Cat-Mu (1250-bp) transposon as a donor DNA. Cat-Mu (or equivalent) is commercially available. Alternatively, custom-made transposons can be constructed in the context of their carrier plasmids, isolated by BglII digestion and purified by anion exchange chromatography as described (Haapa et al., 1999).
    2. Mix ddH2O, glycerol, and 5x complex buffer at RT, and transfer the tube on ice. Take appropriate volumes of the reagents according to the chart below.
    3. Add your donor DNA.
    4. Dilute the MuA transposase in final concentration of 400 ng/μl with cold MuA storage buffer. Dilute only the amount needed. Keep MuA on ice all the time. Pipet MuA up and down before taking an aliquot or adding into the reaction as it is a sticky protein.
    5. Add MuA and transfer the tubes directly into 30 °C water bath. Mix thoroughly!

      Component Amount (μl) Final
      ddH2O 16 Final volume 80 μl
      99.5% glycerol 40 50%
      5x complex buffer 16 1x
      Cat-Mu (1.1 pmol/907.5 ng/μl) 4 4.4 pmol
      MuA transposase (400 ng/μl) 4 1,600 ng
    6. Incubate at 30 °C for 4 h.

  2. Concentrating your complexes
    1. Following assembly, reactions are pooled, and reaction products are concentrated (~10-fold) and desalted using Centricon YM-100 (100-kDa cut off, Millipore) centrifugal cartridges as described (Pajunen et al., 2005). Pooled assembly reactions are diluted up to 2 ml with TGD buffer, applied on the Centricon, centrifuged 30 min 1,000 x g, washed once with 1 ml of TGD buffer (15 min 1,000 x g), and finally recovered with a reverse spin at 300 x g for 2 min. Typical volumes after Centricon concentrators range from 40 to 70 μl.
    2. Alternatively, the concentration step can be done using polyethylene glycol (PEG6000) precipitation as described (Paatero et al., 2008).
    3. Pool your 8 x 80 μl assembly reactions = 640 μl.
    4. (Optional: take 5 μl sample for transposition complex gel)
    5. Add 380 μl filter-sterilized 20% PEG6000 – 2.5 M NaCl. Prepare fresh solution each time you concentrate your complexes.
    6. Incubate (precipitate) on ice 60 min.
    7. (Optional: take 5 μl sample for gel as above)
    8. Centrifuge for 60 min 13,000 rpm at 4 °C in a micro centrifuge.
    9. Discard supernatant carefully without disturbing the pellet.
    10. Resuspend pellet in 50 μl of TGD buffer. The calculated concentration of the Cat-Mu transposition complex preparation is 580.8 ng/μl.
    11. Dissolve the pellet on ice for 60 min or overnight. Tap the tube to help the pellet dissolve.
    12. Preparations are frozen in liquid nitrogen and stored at -80 °C. Transposition complexes can be stored at -80 °C for several months even up to 1 year without loosing activity.
    13. The assembly and concentration of the transposition complexes can be monitored by agarose/heparin/BSA gels as below (see Figure 1).
    14. The transpositional activity of the preparation can be measured by an in vitro transposition assay using plasmid DNA as target and introduction of the reaction products into competent E. coli cells (Haapa et al., 1999). Recovery of chloramphenicol-resistant colonies is an indication of functional transposition complexes.

  3. Transposition complex gel
    1. Prepare an agarose/heparin/BSA gel (2% NuSieve 3: 1 in 1x TAE, 87 μg/ml heparin, 87 μg/ml BSA).
    2. Measure 1 g of NuSieve 3: 1 agarose in a 250-ml flask.
    3. Add 49 ml of ddH2O, and weigh the flask again (should be approx. 49 g).
    4. Melt agarose in microwave oven (or equivalent).
    5. Weigh the flask again, and add H2O so that final weight is again 49 g.
    6. Add 1 ml 50x TAE buffer.
    7. When the solution is about 55 °C, add 43.5 μl of heparin 100 mg/ml and 109 μl of BSA 40 mg/ml.
    8. Prepare gel samples. For non-concentrated complexes (5 μl samples), add 0.5 μl of heparin 65 mg/ml, and 1 μl of 25% Ficoll PM 400 (w/v). For concentrated complexes, take 0.5–1 μl samples; add ddH2O up to 5 μl volume, and then heparin/Ficoll as above.
    9. Run the gel in 1x TAE at 80 V (5 V/cm) for 2 h. Preferably circulate the buffer with a pump.
    10. Stain the gel with ethidium bromide 0.5 μg/ml in 1x TAE for 60 min. Staining can be prolonged up to overnight if necessary.
    11. (Optional: Destain the gel in 1x TAE or ddH2O for 20 min.)
    12. Take a picture (Figure 1).


      Figure 1. A representative transposition complex gel of Em-Mu (1447-bp) complexes (Pajunen et al., 2005). Lane 1, 300 ng of 2-log DNA ladder. Lane 2, 5 μl sample from pooled assembly reaction (250 ng of donor). Lane 3, 10 μl sample from PEG-precipitation before centrifugation (326 ng). Lane 4, 10 μl sample from supernatant. Lane 5, 0.61 μl sample from concentrated complexes dissolved in TGD buffer (410 ng of donor DNA. Lane 6, 1 μl sample from concentrated complexes (665 ng of donor DNA). Lane 7, unassembled control, 410 ng of donor DNA alone. The ca. 3.5 kb band corrensponds to the complex formation between the ends of a single transposon (C1), see Lamberg et al. (2002) for Cat-Mu complex gel.

  4. Preparation of electrocompetent cells (Lamberg et al., 2002)
    1. Inoculate a fresh YeO3-R1 colony into 25 ml SOB medium (without magnesium) in 100 ml flask. Grow the cells overnight with vigorous aeration at 37 °C.
    2. Dilute 0.5 ml of the overnight culture into 500 ml of SOB in a 3 L flask. Grow for 2-3 h with vigorous aeration at 37 °C until the OD600=0.8.
    3. Incubate on ice 30 min.
    4. Divide into three 250 ml bottles. Harvest the cells by centrifugation at 5,000 rpm (2,600 x g) in a Sorvall GSA (or equivalent) rotor for 10 min at 4 °C.
    5. Wash the cell pellet by resuspending each in 5 ml of sterile ice-cold wash buffer (10% glycerol, 90% distilled water, v/v). Keep the bottles on ice at all times.
    6. Combine into two bottles.
    7. Add 200 ml ice-cold wash buffer per bottle, and mix by inverting the bottles several times.
    8. Centrifuge the cell suspension at 5,000 rpm (2,600 x g) for 15 min at 4 °C and carefully pour off the supernatant as soon as the rotor stops. Cells washed in the Wash buffer do not pellet well. If the supernatant is turbid, increase the centrifugation time.
    9. Wash the cell pellet a second time by resuspending in 5 ml of sterile ice-cold wash buffer. Add 200 ml wash buffer, and mix by inverting the bottle several times.
    10. Centrifuge the cell suspension at 5,000 rpm for 15 min.
    11. Pour off the supernatant and resuspend the cells in the Wash buffer that remains in the centrifuge bottle. Combine into single bottle. Final volume is approximately 2 ml (~1x1011 cells/ml).
    12. Incubate on ice at least 30 min, preferably 60 min.
    13. Cells can be used immediately or can be frozen in aliquots. For some strains like YeO3-R1 fresh cells yield better efficiencies.
    14. For freezing divide 60 μl (or 120 μl) in sterile cold micro centrifuge tubes. Flash-freeze in liquid nitrogen and store at -80 °C.

  5. Preparation transposon insertion library (Pinta et al., 2012)
    1. To generate the library, 8 individual electroporations are performed as follows.
    2. Take 50 μl of freshly-prepared electrocompetent YeO3-R1 cells. Mix on ice with 1.7 μl (990 ng; preferentially use 800-1,000 ng of complexes for highest efficiency) of Cat-Mu transposition complex preparation.
    3. Transfer the mixture to a pre-chilled 0.1-cm electrode spacing cuvette, and carry out electroporation using Genepulser II with the following settings: 200 Ω, 1.8 kV, 25 μF.
    4. Add 1 ml of pre-warmed SOC medium, and combine the suspensions from the individual electroporations in a 50-ml flask.
    5. Grow bacteria for 50 min at 28 °C with agitation (220 rpm).
    6. Add 3.3 ml 50% glycerol to the final concentration of 15% (v/v).
    7. Freeze the library as aliquots (e.g. 14 x 0.8-ml) in liquid nitrogen, and store at -80 °C.
    8. To estimate the number of individual transposon insertion mutants in the library thaw one aliquot and spread on LA plates with Chloramphenicol (10 μg/ml) (LA-Clm).
    9. Incubate 1–2 days at 28 °C (i.e., optimal growth temperature for Y. enterocolitica), and calculate the number of colony forming units (CFU).

  6. Isolation of enterocoliticin resistant mutants (Pinta et al., 2012)
    1. From the library aliquots prepared above spread a total of 16,000 CFU on LA-Clm plates such that you obtain individual colonies. Typically spread four library aliquots as 50 μl aliquots to 40-50 LA-Clm plates. 
    2. Incubate 2 days at RT.
    3. Pool the Clm-resistant colonies by adding 1 ml PBS / plate and detach the colonies gently by scraping with an L-spreader.
    4. With a pipette transfer the PBS-suspension into a 50 ml Falcon tube.
    5. Pool the colonies from all other plates and combine them into one 50 ml tube. Mix the tube gently but thoroughly f. ex. by pulse vortexing.
    6. To prepare the pooled transposon library for long-time storage (i.e., the part you don't use immediately) follow steps 8-10.
    7. To continue with the isolation of enterocoliticin resistant mutants follow the steps from step 11 forward.
    8. Centrifuge the 50 ml tube at 3,000 rpm (2,000 x g) for 15 min at 4 °C and carefully remove most of the supernatant.
    9. Resuspend the pellet in 5 ml of 15% glycerol in TSB.
    10. Freeze the library as 0.5 ml aliquots and store at -80 °C.
    11. Measure the OD600 of the pooled transposon library from a suitable dilution (f.ex. 1:100).
    12. Based on the result dilute the cell suspension with PBS to OD600 ~0.2.
    13. Mix 10 μl of the OD600 ~0.2 suspension with 10 μl of enterocoliticin (Strauch et al., 2001) (suitable enterocoliticin-dose is determined in section VII) and 80 μl of TSB.
    14. Spread the mixture on a LA-Clm plate.
    15. Repeat steps 13-14 four times to get altogether 5 plates.
    16. Incubate the plates 1-2 days at RT to allow the growth of enterocoliticin-resistant mutants.
    17. Pick up single colonies from the plates and spread as pure cultures on new LA-Clm plates. These will constitute the candidate enterocoliticin-resistant mutants.
    18. Incubate the plates 1-2 days at RT.
    19. The enterocoliticin-resistance of the pure cultures has to be confirmed using a drop-test. Divide an LA-Clm plate into 8-12 sectors and culture the mutants on the sectors as a lawn using inoculation loops. As control, culture known enterocoliticin-sensitive and –resistant bacteria on some sectors.
    20. Immediately, apply a 2 μl drop of enterocoliticin in the center of each lawn.
    21. Incubate the plate 1-2 days at RT and inspect the sectors.
    22. he enterocoliticin-sensitive bacteria have no growth on the spot where enterocoliticin was applied while the enterocoliticin-resistant bacteria grow all over the sector. 
      Continue with the enterocoliticin-resistant mutants to section 8.

  7. Determination of the killing potential of the enterocoliticin solution
    1. Use a fresh colony of YeO3-R1 to inoculate 5 ml of TSB in a 15 ml tube. Grow the cells overnight with aeration at RT.
    2. Measure the OD600 of the culture. Dilute the culture in PBS so to OD600 ~0.2.
    3. Mix 10 μl of the OD600 ~0.2 suspension with different amounts of the enterocoliticin solution [Strauch et al., 2001] (f. ex. 1, 5, 10, 30 and 50 μl). Fill up the volume of the mixtures to 100 μl with TSB.
    4. Plate the different mixtures on separate LA-Clm plates.
    5. Incubate the plates 1-2 days at RT.
    6. Count the spontaneous resistant colonies. There should be less than ten spontaneous resistant colonies / plate.

  8. Identification of the transposon insertion site
    The Cat-Mu transposon insertion sites can be identified by sequencing the transposon-containing genomic fragment that are cloned into pUC19 (Pinta et al., 2012).
    1. For the cloning of the transposon insertion fragment, HindIII digested genomic DNA (isolated using a DNA-isolation kit suitable for bacteria) of the mutant is cloned into HindIII digested pUC19 using standard protocols. HindIII does not cleave the Cat-Mu, thus ClmR transformants can be selected.
    2. The inserts in the plasmids isolated from the transformants can be sequenced using transposon-specific primers Muc1 and Muc2 (5'-GCTCTCCCCGTGGAGGTAAT-3' and 5'-TTCCGTCACAGGTATTTATTCGGT-3', respectively). The sequences obtained from these allow identification of the Cat-Mu -flanking regions on both sides of the transposon-insertions.

Recipes

  1. TGD buffer
    10 mM Tris-HCl (pH 6.0)
    0.5% glycerol
    0.1 mM DTT
  2. 5x complex buffer (10 ml)
    3,750 μl 2 M Tris-HCl (pH 6)
    125 μl 10% Triton X-100
    2,500 μl 3 M NaCl
    10 μl 0.5 M EDTA (pH 8)
    3,615 μl ddH2O
    Filter-sterilize (0.2 μm) and divide into aliquots.
    Can be stored at -20 °C for 1 year
  3. 1 M DTT (20 ml)
    3.09 g DTT
    Add 0.01 M Sodium acetate (NaAc) pH 5.2 to final volume.
    Filter-sterilize (0.2 μm) and divide into aliquots.
    Can be stored at -20 °C for 1 year.
  4. 20% PEG6000 (w/v) - 2.5 M NaCl (5 ml)
    1 g PEG6000
    2.5 ml 5 M NaCl
    Add ddH2O to final volume.
    Filter-sterilize (0.2 μm) and use during the same day
  5. SOB medium (1 L) without magnesium
    20 g Bacto-tryptone
    5 g Bacto-yeast extract
    0.584 g NaCl
    0.186 g KCl
    Adjust pH to 7.0 with 5 M NaOH
    Add ddH2O to final volume and autoclave
    Stored at RT for up to 1 month
  6. 2 M Mg++ stock
    20.33 g MgCl2 · 6H2O
    24.65 g MgSO4 · 7H2O
    Add ddH2O to final volume.
    Filter-sterilize (0.2 μm) or autoclave.
    Stored at 4 °C for up to 6 months
  7. 2 M glucose (100 ml)
    26.04 g glucose
    Add ddH2O to final volume
    Filter-sterilize (0.2 μm)
    Stored at 4 °C for up to 6 months
  8. SOC medium (100 ml)
    1 ml 2 M Mg++ stock
    1 ml 2 M glucose
    98 ml SOB medium without magnesium
  9. Luria broth (LB) medium (1 L)
    10 g Bacto-tryptone
    5 g Bacto-yeast extract
    5 g NaCl
    Adjust pH to 7.0 with 5 M NaOH
    Add ddH2O to final volume and autoclave
    Stored at RT for up to 1 month
  10. LB agar (LA) plates (1 L)
    1 L LB medium
    15 g Bacto-agar
    Add Bacto-agar to LB medium before autoclaving
    Cool down to 55 °C
    Supplement with suitable antibiotics and dispense approx. 20 ml per Petri dish
    Stored at 4 °C for up to 1 month
  11. Chloramphenicol (Clm) solution (10 ml)
    0.2 g Chloramphenicol
    Add absolute ethanol to final volume
    Stock solution (20 mg/ml) can be stored at –20 °C for 1 year
    Chloramphenicol is used at the final concentration of 10 μg/ml
  12. Tryptic soya broth
    Prepare as instructed by the manufacturer
  13. Phosphate-buffered saline (PBS) (pH 7.4) (1 L)
    Prepare 1 L of 1x PBS as follows. Add to 800 ml of ddH2O 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4. Adjust the pH to 7.4 with HCl. Add distilled water to a total volume of 1 L. Dispense the solution into aliquots and sterilize by autoclaving (20 min, 121 °C, liquid cycle). Stored at RT.
  14. TAE buffer
    2 M Tris-acetate (pH 8.0)
    50 mM EDTA

Acknowledgments

The protocol was adapted from our previously published paper Pinta et al. (2012). This work was supported by the Academy of Finland (projects 114075, 104361 and 50441) to M.S. and Finnish Glycoscience Graduate School to E.P. We thank Dr Eckhard Strauch for providing the enterocoliticin used in the work.

References

  1. al-Hendy, A., Toivanen, P. and Skurnik, M. (1992). Lipopolysaccharide O side chain of Yersinia enterocolitica O:3 is an essential virulence factor in an orally infected murine model. Infect Immun 60(3): 870-875.
  2. Haapa, S., Taira, S., Heikkinen, E. and Savilahti, H. (1999). An efficient and accurate integration of mini-Mu transposons in vitro: a general methodology for functional genetic analysis and molecular biology applications. Nucleic Acids Res 27(13): 2777-2784.
  3. Lamberg, A., Nieminen, S., Qiao, M. and Savilahti, H. (2002). Efficient insertion mutagenesis strategy for bacterial genomes involving electroporation of in vitro-assembled DNA transposition complexes of bacteriophage mu. Appl Environ Microbiol 68(2): 705-712.
  4. Pajunen, M. I., Pulliainen, A. T., Finne, J. and Savilahti, H. (2005). Generation of transposon insertion mutant libraries for Gram-positive bacteria by electroporation of phage Mu DNA transposition complexes. Microbiology 151(Pt 4): 1209-1218.
  5. Paatero, A. O., Turakainen, H., Happonen, L. J., Olsson, C., Palomaki, T., Pajunen, M. I., Meng, X., Otonkoski, T., Tuuri, T., Berry, C., Malani, N., Frilander, M. J., Bushman, F. D. and Savilahti, H. (2008). Bacteriophage Mu integration in yeast and mammalian genomes. Nucleic Acids Res 36(22): e148.
  6. Pinta, E., Li, Z., Batzilla, J., Pajunen, M., Kasanen, T., Rabsztyn, K., Rakin, A. and Skurnik, M. (2012). Identification of three oligo-/polysaccharide-specific ligases in Yersinia enterocolitica. Mol Microbiol 83(1): 125-136.
  7. Strauch, E., Kaspar, H., Schaudinn, C., Dersch, P., Madela, K., Gewinner, C., Hertwig, S., Wecke, J. and Appel, B. (2001). Characterization of enterocoliticin, a phage tail-like bacteriocin, and its effect on pathogenic Yersinia enterocolitica strains. Appl Environ Microbiol 67(12): 5634-5642.

简介

串联亲和纯化(TAP)(Pugi等人,2001; Rigaut等人,1999)是使用目标靶蛋白的标记方法的方法两步纯化方案以在天然条件和表达水平下下拉蛋白复合物。 TAP标签由三种组分组成:钙调素结合肽,烟草蚀纹病毒(TEV)蛋白酶切割位点和作为免疫球蛋白G(IgG)结合结构域的蛋白A.该方案从酵母细胞中使用的原始方法(Pugi等人,2001; Rigaut等人,1999)修饰,用于从果蝇头分离蛋白质复合物,以及卵巢表达感兴趣的TAP标记的蛋白质。为了确定果蝇脆弱X蛋白(dFMR1)的体内结合伴侣,我们开发了表达重组形式的具有羧基末端TAP标签的dFMR1的苍蝇的转基因菌株(Tsai和Carstens,2006)。为了确保构建体在野生型水平表达,我们在救援突变不育表型的基因组拯救构建体的上下文中工程化这种形式的标记蛋白质。使用温和条件进行纯化过程以维持天然蛋白质相互作用。对于在果蝇S2细胞培养物中的TAP方法,我们成功地使用了由Tsai和Carstens先前公布的方案(Tsai和Carstens,2006; Bhogal等人,2011)。...

关键字:小肠结肠炎耶尔森菌, Mu转座, 转座子插入库, 脂多糖, 噬菌体

材料和试剂

  1. 小肠结肠炎耶尔森菌菌株YeO3-R1(al-Hendy等人,1992)
  2. Cat-Mu转座子(Haapa等人,1999)或等同物如Entranceposon Cam-R3(Thermo Fisher Scientific,Finnzymes,目录号:F-778)
  3. MuA转座酶1,100ng /μl和MuA储存缓冲液(Thermo Fisher Scientific,Finnzymes,目录号:F-750C)
  4. 99.5%甘油BD H(目录号: 24388.320)
  5. 10%Triton X-100(w/v)(F.Hoffmann-La Roche,目录号:11332481001)
  6. 超纯0.5M EDTA(pH 8.0)(Life Technologies,Invitrogen TM,目录号:15575)
  7. DTT(Sigma-Aldrich,目录号:D0632)
  8. Centricon YM-100(100-kDa cutt off)(EMD Millipore,目录号:UFC210024PL)
  9. 聚乙二醇(PEG)6000(Merck KGaA,目录号:8.07491)
  10. NuSieve 3:1琼脂糖(25g)(Lonza,目录号:50091)
  11. 来自牛血清,BSA(Sigma-Aldrich,目录号:A7906)的白蛋白,
  12. 肝素(Sigma-Aldrich,目录号:H3393)
  13. Ficoll PM 400(Sigma-Aldrich,目录号:F4375)
  14. 细菌用琼脂,细菌用胰蛋白胨和细菌用酵母提取物(BD Difco)
  15. 氯霉素(Sigma-Aldrich,目录号:C0378)
  16. 胰蛋白酶大豆肉汤(Oxoid Limited,目录号:CM0129)
  17. 由Y产生的肠念珠菌。 肠结肠炎血清型O:7,8株29930(Strauch等人,2001)
  18. MuA存储缓冲区
  19. 醋酸钠(NaAc)
  20. DNA梯度(New England Biolabs)
  21. 磷酸盐缓冲盐水(PBS)(pH 7.4)
  22. TGD缓冲区(参见配方)
  23. 5x复合缓冲液(见配方)
  24. 1 M DTT(见配方)
  25. 20%PEG6000(w/v)-2.5M NaCl(5ml)(参见配方)
  26. 不含镁的SOB培养基(1L)(参见配方)
  27. 2 M Mg ++原料(见配方)
  28. 2 M葡萄糖(100 ml)(见配方)
  29. SOC培养基(100ml)(参见配方)
  30. Luria肉汤(LB)培养基(1L)(见Recipes)
  31. LB琼脂(LA)平板(1L)(参见配方)
  32. 氯霉素(Clm)溶液(10ml)(参见配方)
  33. 胰蛋白酶大豆肉汤(见Recipes)
  34. 磷酸盐缓冲盐水(PBS)(pH 7.4)(1L)(参见Recipes)
  35. TAE缓冲区(请参阅配方)

设备

  1. 离心机(Sorvall,Heraeus Holding)
  2. 水浴(格兰特)
  3. 孵化器(Termaks)
  4. 摇床(New Brunswick Scientific)
  5. 生物测定仪(Eppendorf)
  6. 琼脂糖凝胶电泳(Bio-Rad Laboratories)
  7. 凝胶文件设备(Bio-Rad Laboratories)
  8. 电穿孔仪(Bio-Rad Laboratories,Genepulser II)
  9. 90mm培养皿(Thermo Fisher Scientific,Sterilin ,目录号:101RT)
  10. 0.1-cm电穿孔杯(Bio-Rad Laboratories)

程序

  1. 转置复合体装配(Lamberg等人,2002)
    1. 使用Cat-Mu(1250-bp)转座子作为供体DNA制备八个标准装配反应。 Cat-Mu(或等价物)是可商购的。或者,可以在其载体质粒的上下文中构建定制的转座子,通过BglII消化分离并如所述通过阴离子交换层析纯化(Haapa等人,1999)。
    2. 在室温下混合ddH 2 O,甘油和5x复合缓冲液,并在冰上转移管。根据下表采取适当体积的试剂。
    3. 添加您的供体DNA。
    4. 用冷MuA储存缓冲液稀释最终浓度为400 ng /μl的MuA转座酶。只稀释所需的量。保持MuA一直在冰上。 Pipet MuA上下取下等分试样或加入反应中,因为它是一种粘性蛋白质。
    5. 添加MuA,并将管直接转移到30°C水浴。彻底混合!

      =""border ="1"bordercolor ="#000000"cellpadding ="0"cellspacing ="0">
      零件 量(μl) 最后
      ddH 2 O 16 最终体积80μl
      99.5%甘油 40 50%
      5x复合缓冲液 16 1x
      Cat-Mu(1.1pmol/907.5ng /μl) 4 4.4pmol
      MuA转座酶(400ng /μl) 4 1,600ng
    6. 在30℃孵育4小时。

  2. 集中你的复合物
    1. 组装后,汇集反应,并且如所述(Pajunen等人,2005)使用Centricon YM-100(100-kDa截留,Millipore)离心筒将反应产物浓缩(〜10倍)并脱盐。将汇集的装配反应用TGD缓冲液稀释至2ml,施加在Centricon上,离心30分钟1000×g,用1ml TGD缓冲液洗涤一次(15分钟1,000×g ),最后用300×g的反向旋转回收2分钟。 Centricon浓缩器后的典型体积为40至70μl。
    2. 或者,可以使用如(Paatero等人,2008)所述的聚乙二醇(PEG6000)沉淀进行浓缩步骤。
    3. 池8 x 80微升组装反应= 640微升。
    4. (可选:取5μl转移复合凝胶样品)
    5. 加入380μl过滤灭菌的20%PEG6000 - 2.5 M NaCl。每次你集中你的复合体准备新鲜的解决方案。
    6. 在冰上孵育(沉淀)60分钟。
    7. (可选:取5μl样品用于凝胶,如上)
    8. 在微量离心机中在4℃下离心60分钟13,000rpm。
    9. 小心弃去上清液,不影响沉淀。
    10. 重悬在50μlTGD缓冲液中的沉淀。 Cat-Mu转座复合物制剂的计算浓度为580.8ng /μl
    11. 将颗粒在冰上溶解60分钟或过夜。 点击管以帮助丸粒溶解。
    12. 将制备物在液氮中冷冻并储存在-80℃。置换复合物可以在-80℃下储存几个月,甚至长达1年,而不会失去活性
    13. 可以通过如下的琼脂糖/肝素/BSA凝胶监测转座复合物的装配和浓度(参见图1)。
    14. 制剂的转位活性可以通过使用质粒DNA作为靶的体外转座试验和将反应产物引入感受态细胞中来测量。大肠杆菌细胞(Haapa等人,1999)。回收氯霉素抗性菌落是功能性转座复合体的指示
  3. 转位复合凝胶
    1. 制备琼脂糖/肝素/BSA凝胶(2x TAE中的2%NuSieve 3:1,87μg/ml肝素,87μg/ml BSA)。
    2. 在250-ml烧瓶中测量1g NuSieve 3:1琼脂糖。
    3. 加入49ml ddH 2 O,并再次称重烧瓶(应为约49g)。
    4. 在微波炉中熔化琼脂糖(或等同物)
    5. 再次称重烧瓶,加入H 2 O,使最终重量为49g。
    6. 加入1ml 50x TAE缓冲液。
    7. 当溶液为约55℃时,加入43.5μl的肝素100mg/ml和109μl的BSA 40mg/ml。
    8. 制备凝胶样品。对于非浓缩复合物(5μl样品),加入0.5μl肝素65mg/ml和1μl25%Ficoll PM 400(w/v)。对于浓缩复合物,取0.5-1μl样品;加入ddH 2 O 2至5μl体积,然后加入肝素/Ficoll,如上所述。
    9. 在1X TAE中在80V(5V/cm)下运行凝胶2小时。最好用泵循环缓冲液。
    10. 用溴化乙锭0.5μg/ml在1x TAE中凝胶化凝胶60分钟。如有必要,染色可以延长一夜。
    11. (可选:将凝胶在1x TAE或ddH 2 O中脱色20分钟)
    12. 拍摄照片(图1)。


      图1. Em-Mu(1447-bp)复合物的代表性转座复合物凝胶(Pajunen等,2005)。泳道1,300ng的2 -log DNA梯。泳道2,5μl来自汇集装配反应的样品(250ng供体)。泳道3,10μl来自离心前PEG沉淀的样品(326ng)。泳道4,10μl来自上清液的样品。泳道5,0.61μl来自溶解在TGD缓冲液中的浓缩复合物的样品(410ng供体DNA,泳道6,1μl 来自浓缩复合物(665ng供体DNA)的样品。 泳道7,未组装的对照,仅410ng供体DNA。 大约。 3.5kb带响应于单个转座子(C1)末端之间的复合物形成,参见Lamberg等人(2002)对于Cat-Mu复合物凝胶。

  4. 电感受态细胞的制备(Lamberg等人,2002)
    1. 在100ml烧瓶中将新鲜的YeO3-R1菌落接种到25ml SOB培养基(不含镁)中。 在37℃下剧烈通气使细胞生长过夜。
    2. 在3L烧瓶中将0.5ml过夜培养物稀释到500ml SOB中。 生长2-3小时,在37℃剧烈通气直到OD 600 = 0.8。
    3. 在冰上孵育30分钟。
    4. 分成三个250毫升瓶。 通过在Sorvall GSA(或等价物)转子中在5,000rpm(2,600xg)下在4℃下离心10分钟收获细胞。
    5. 通过将每个重悬于5ml无菌冰冷的洗涤缓冲液(10%甘油,90%蒸馏水,v/v)中来洗涤细胞沉淀。 保持瓶子在冰上的所有时间。
    6. 合并成两瓶。
    7. 每瓶加入200毫升冰冷的洗涤缓冲液,倒置瓶子几次混合
    8. 在4℃下以5,000rpm(2,600×g)离心细胞悬浮液15分钟,并在转子停止时小心地倒出上清液。 在洗涤缓冲液中洗涤的细胞不会沉淀。 如果上清液浑浊,请增加离心时间
    9. 通过重悬于5ml无菌冰冷的洗涤缓冲液中,再次洗涤细胞沉淀。 加入200毫升洗涤缓冲液,倒置瓶子几次混合。
    10. 以5,000 rpm离心细胞悬浮液15分钟。
    11. 倒出上清液,并将细胞悬浮在留在离心瓶中的洗涤缓冲液中。 组合成单瓶。 最终体积为约2ml(〜1×10 11细胞/ml)。
    12. 在冰上孵育至少30分钟,优选60分钟。
    13. 细胞可以立即使用或可以等分冷冻。对于一些菌株,如YeO3-R1,新鲜细胞产生更好的效率。
    14. 对于冷冻分离60微升(或120微升)在无菌冷微量离心管中。 在液氮中快速冷冻并储存在-80℃。

  5. 制备转座子插入文库(Pinta等人,2012)
    1. 为了产生文库,如下进行8次个别电穿孔。
    2. 取50μl新鲜制备的electrocompetent YeO3-R1细胞。 在冰上与1.7μl(990 ng;优先使用800 - 1,000 ng的配合物,以获得最高效率)的Cat-Mu转座复合物制剂混合。
    3. 将混合物转移到预冷的0.1厘米电极间距比色皿,并使用Genepulser II进行电穿孔,具有以下设置:200Ω,1.8kV,25μF。
    4. 加入1ml预热的SOC培养基,并将来自单个电穿孔的悬浮液在50ml烧瓶中混合。
    5. 在28℃下搅拌(220rpm)生长细菌50分钟。
    6. 加入3.3ml 50%甘油至15%(v/v)的终浓度。
    7. 将该文库作为等分试样(例如14x0.8ml)在液氮中冷冻,并储存在-80℃。
    8. 为了估计文库中单个转座子插入突变体的数目,将一个等分试样解冻,并用氯霉素(10μg/ml)(LA-Clm)铺在LA板上。
    9. 在28℃孵育1-2天(即,肠炎沙门氏菌的最佳生长温度),并计算菌落形成单位数(CFU)。

  6. 肠球菌素抗性突变体的分离(Pinta等,2012)
    1. 从上述制备的文库中,在LA-Clm平板上铺满总共16,000CFU,使得获得单个菌落。 通常将四个文库等分试样作为50μl等分试样分装到40-50LA-Clm平板上。
    2. 在室温下孵育2天。
    3. 通过加入1ml PBS /板来汇集Clm抗性菌落,并通过用L-spreader刮擦轻轻地分离集落。
    4. 用移液管将PBS-悬浮液转移到50ml Falcon管中。
    5. 从所有其他板集中殖民地,并将它们结合成一个50毫升管。 轻轻地,但彻底地混合管。 例如。 通过脉冲涡流
    6. 要准备汇集的转座子库以进行长期存储(即不立即使用的部分),请按照步骤8-10操作。
    7. 继续分离肠炎小体抗性突变体遵循从步骤11向前的步骤。
    8. 在4℃下,以3,000rpm(2,000xg)离心50ml管15分钟,小心地除去大部分上清液。
    9. 将沉淀重悬于5ml TSP中的15%甘油中。
    10. 将该库冻结成0.5ml等分试样并储存在-80℃。
    11. 从合适的稀释液(f.ex. 1:100)测量合并的转座子文库的OD 600。
    12. 基于结果,将细胞悬浮液用PBS稀释至OD 600〜0.2
    13. 将10μlOD 600〜0.2悬浮液与10μl肠道粘蛋白混合(Strauch等人,2001)(合适的肠溶胆素剂量在VII部分中测定)和80μl μl的TSB
    14. 将混合物铺在LA-Clm板上。
    15. 重复步骤13-14四次,共5板。
    16. 在室温孵育平板1-2天,以允许肠炎支原体抗性突变体的生长
    17. 从板中取出单个菌落并作为纯培养物铺在新的LA-Clm板上。这些将构成候选肠球菌素抗性突变体。
    18. 在室温下孵育平板1-2天。
    19. 纯培养物的肠球菌组织抗性必须使用滴试验证实。将LA-Clm板分为8-12个部分,并使用接种环将培养突变体作为草坪的部分。作为对照,在某些部门培养已知的肠球菌敏感和抗性细菌
    20. 立即,在每个草坪的中心,应用2微升enterocoliticin滴。
    21. 在室温下孵育1-2天,并检查扇区
    22. 他对肠道小杆菌素敏感的细菌在应用肠炎杆菌素的地方没有生长,而肠炎支原体抗性细菌在整个区域生长。 
      继续第8节的肠炎支原体抗性突变体。

  7. 确定肠道凝胶溶液的杀伤电位
    1. 使用新鲜的YeO3-R1殖民地接种15毫升管中的5毫升TSB。 在室温下通气培养细胞过夜。
    2. 测量培养物的OD 600。 在PBS中稀释培养物至OD 600〜0.2。
    3. 将10μl的OD 600〜0.2悬浮液与不同量的肠炎支原体溶液混合[Strauch et al。,2001](f.1,5,10, 30和50μl)。 使用TSB填充混合物的体积至100μl。
    4. 将不同的混合物铺在单独的LA-Clm板上。
    5. 在室温下孵育平板1-2天。
    6. 计数自发抗性集落。 应该有少于10个自发抗性菌落/板
  8. 鉴定转座子插入位点
    Cat-Mu转座子插入位点可以通过对克隆到pUC19中的含转座子的基因组片段进行测序来鉴定(Pinta等人,2012)。
    1. 为了克隆转座子插入片段,使用标准方案将HindIII消化的基因组DNA(使用适用于细菌的DNA分离试剂盒分离)克隆到HindIII消化的pUC19中。 HindIII不切割Cat-Mu,因此可以选择ClmR转化体。
    2. 可以使用转座子特异性引物Muc1和Muc2(分别为5'-GCTCTCCCCGTGGAGGTAAT-3'和5'-TTCCGTCACAGGTATTTATTCGGT-3')对从转化体分离的质粒中的插入片段进行测序。 从这些序列获得允许鉴定转座子插入两侧的Cat-Mu侧翼区。

食谱

  1. TGD缓冲区
    10mM Tris-HCl(pH6.0) 0.5%甘油 0.1 mM DTT
  2. 5x复合缓冲液(10ml) 3,750μl2 M Tris-HCl(pH 6)
    125μl10%Triton X-100 2,500μl3 M NaCl
    10μl0.5M EDTA(pH8)
    3,615μlddH 2 O 2/b 过滤灭菌(0.2μm)并分成等份 可存储于-20年°C 一年
  3. 1 M DTT(20ml) 3.09克DTT
    加入0.01M乙酸钠(NaAc),pH 5.2至最终体积 过滤灭菌(0.2μm)并分成等份 可在-20°C储存1年。
  4. 20%PEG6000(w/v)-2.5M NaCl(5ml) 1 g PEG6000
    2.5ml 5M NaCl
    将ddH2O加入最终体积。
    过滤灭菌(0.2μm)并在同一天使用
  5. SOB介质(1L),不含镁
    20克细菌用胰蛋白胨 5克细菌 - 酵母提取物
    0.584g NaCl
    0.186克KCl
    用5 M NaOH将pH调节至7.0 将ddH 2 O加入到最终体积并高压灭菌
    在室温储存长达1个月
  6. 2 M Mg ++股
    20.33g MgCl 2·6H 2 O
    24.65g MgSO 4·7H 2 O
    将ddH 2 O添加到最终体积中。
    过滤灭菌(0.2μm)或高压灭菌 储存在4°C长达6个月
  7. 2 M葡萄糖(100ml)
    26.04克葡萄糖 将ddH 2 O添加到最终量
    过滤灭菌(0.2μm)
    储存在4°C长达6个月
  8. SOC培养基(100ml)
    1ml 2M Mg ++储液
    1 ml 2 M葡萄糖
    98毫升不含镁的SOB培养基
  9. Luria肉汤(LB)培养基(1L) 10克细菌用胰蛋白胨 5克细菌 - 酵母提取物
    5克NaCl
    用5 M NaOH将pH调节至7.0 将ddH 2 O加入到最终体积并高压灭菌
    在室温储存长达1个月
  10. LB琼脂(LA)平板(1L) 1 L LB培养基
    15克细菌琼脂 在高压灭菌前向LB培养基中加入Bacto琼脂
    冷却至55°C
    补充适当的抗生素和分配约。 每个培养皿中加入20ml 储存于4°C长达1个月
  11. 氯霉素(Clm)溶液(10ml) 0.2克氯霉素
    加入无水乙醇至最终体积
    储备溶液(20mg/ml)可以在-20℃下储存1年
    氯霉素以10μg/ml的终浓度使用
  12. 胰蛋白酶大豆培养基
    根据制造商的指示进行准备
  13. 磷酸盐缓冲盐水(PBS)(pH 7.4)(1L)
    准备1 L 1×PBS如下。 加入到800ml ddH 2 O 8中的8g NaCl,0.2g KCl,1.44g Na 2 HPO 4和0.24g 的KH 2 PO 4 4。 用HCl调节pH至7.4。 加入蒸馏水至总体积为1L。将溶液分成等份并通过高压灭菌(20分钟,121℃,液体循环)灭菌。 储存在RT。
  14. TAE缓冲区
    2 M Tris-乙酸盐(pH 8.0)
    50mM EDTA

致谢

该协议改编自我们先前发表的论文Pinta等人(2012)。这项工作得到了芬兰科学院(项目114075,104361和50441)的支持。和芬兰糖科学研究生院。我们感谢Eckhard Strauch博士提供在工作中使用的肠球菌肽。

参考文献

  1. Al-Hendy,A.,Toivanen,P。和Skurnik,M。(1992)。 小肠结肠炎耶尔森菌O:3的脂多糖O侧链是口服感染的小鼠模型中的必需毒力因子。 Infect Immun 60(3):870-875。
  2. Haapa,S.,Taira,S.,Heikkinen,E。和Savilahti,H。(1999)。 微型Mu转座子在体外的高效精确整合:a用于功能遗传分析和分子生物学应用的一般方法。核酸研究 27(13):2777-2784。
  3. Lamberg,A.,Nieminen,S.,Qiao,M。和Savilahti,H。(2002)。 涉及体外电穿孔的细菌基因组的有效插入诱变策略 - 组装DNA transposition complexes of bacteriophage mu。 Appl emviron Microbiol 68(2):705-712。
  4. Pajunen,M.I.,Pulliainen,A.T.,Finne,J。和Savilahti,H。(2005)。 通过电穿孔噬菌体Mu DNA转座复合物产生革兰氏阳性细菌的转座子插入突变体文库。 Microbiology 151(Pt 4):1209-1218。
  5. Paatero,AO,Turakainen,H.,Happonen,LJ,Olsson,C.,Palomaki,T.,Pajunen,MI,Meng,X.,Otonkoski,T.,Tuuri,T.,Berry,C.,Malani,N 。,Frilander,MJ,Bushman,FDand Savilahti,H。(2008)。 噬菌体Mu在酵母和哺乳动物基因组中的整合。核酸研究 em> 36(22):e148。
  6. Pinta,E.,Li,Z.,Batzilla,J.,Pajunen,M.,Kasanen,T.,Rabsztyn,K.,Rakin,A.and Skurnik, 在肠结肠炎耶尔森菌中鉴定三种寡糖/多糖特异性连接酶/a>。 Mol Microbiol 83(1):125-136。
  7. Strauch,E.,Kaspar,H.,Schaudinn,C.,Dersch,P.,Madela,K.,Gewinner,C.,Hertwig,S.,Wecke,J.and Appel, 肠炎杆菌素,噬菌体尾样细菌素的表征及其对致病性肠炎耶尔森氏菌的影响 应用环境微生物 67(12):5634-5642。
  • 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: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Pajunen, M., Pinta, E. and Skurnik, M. (2012). Construction and Screening of a Transposon Insertion Library of Yersinia enterocolitica (YeO3-R1). Bio-protocol 2(15): e246. DOI: 10.21769/BioProtoc.246.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。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或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。