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Expression and Purification of the Cas10-Csm Complex from Staphylococci
表达并纯化葡萄球菌的Cas10-Csm复合体   

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Journal of Bacteriology
Jan 2014

Abstract

CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a class of prokaryotic immune systems that degrade foreign nucleic acids in a sequence-specific manner. These systems rely upon ribonucleoprotein complexes composed of Cas nucleases and small CRISPR RNAs (crRNAs). Staphylococcus epidermidis and Staphylococcus aureus are bacterial residents on human skin that are also leading causes of antibiotic resistant infections (Lowy, 1998; National Nosocomial Infections Surveillance, 2004; Otto, 2009). Many staphylococci possess Type III-A CRISPR-Cas systems (Marraffini and Sontheimer, 2008; Cao et al., 2016), which have been shown to prevent plasmid transfer and protect against viral predators (Goldberg et al., 2014; Hatoum-Aslan et al., 2014; Samai et al., 2015) in these organisms. Thus, gaining a mechanistic understanding of these systems in the native staphylococcal background can lead to important insights into the factors that impact the evolution and survival of these pathogens. Type III-A CRISPR-Cas systems encode a five-subunit effector complex called Cas10-Csm (Hatoum-Aslan et al., 2013). Here, we describe a protocol for the expression and purification of Cas10-Csm from its native S. epidermidis background or a heterologous S. aureus background. The method consists of a two-step purification protocol involving Ni2+-affinity chromatography and a DNA affinity biotin pull-down, which together yield a pure preparation of the Cas10-Csm complex. This approach has been used previously to analyze the effects of mutations on Cas10-Csm complex integrity (Hatoum-Aslan et al., 2014), crRNA formation (Hatoum-Aslan et al., 2013), and to detect binding partners that directly interact with the core Cas10-Csm complex (Walker et al., 2016). Importantly, this approach can be easily adapted for use in other Staphylococcus species to probe and understand their native Type III-A CRISPR-Cas systems.

Keywords: CRISPR-Cas Type III-A (CRISPR-Cas Type III-A), Cas10-Csm ( Cas10-Csm), Staphylococci ( 葡萄球菌), Protein purification ( 蛋白质纯化), Protein Expression ( 蛋白质表达), DNA affinity chromatography ( DNA亲和层析), Biotin pull-down ( 生物素 pull-down)

Background

Staphylococcus epidermidis and Staphylococcus aureus are prevalent skin-dwelling bacteria that have a range of opposing impacts. While S. aureus asymptomatically colonizes ~30% of the population (Conlan et al., 2012), this organism is a leading cause of skin and soft tissue infections (Stryjewski and Chambers, 2008; Grice and Segre, 2011). In contrast, S. epidermidis is generally considered beneficial, and promotes human health by 1) preventing S. aureus colonization (Iwase et al., 2010), 2) producing antimicrobial peptides that target skin pathogens (Cogen et al., 2010), and 3) stimulating the human immune system to facilitate pathogen defense (Lai et al., 2010; Naik et al., 2015). However, when allowed to breach the skin barrier, this species can also cause antibiotic resistant infections, particularly on indwelling medical devices (Otto, 2009; Harris and Richards, 2006). Furthermore, pathogenic staphylococci that are resistant to all known antibiotics have recently emerged in both hospital and community settings (Furuya and Lowy, 2006) and have become a major threat to global public health. Horizontal gene transfer (HGT), or the exchange of genetic information between related bacterial species, is a major route by which these organisms acquire virulence factors and multi-drug resistance. Therefore, it is of utmost importance to understand the factors that impact and regulate HGT in these organisms.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated proteins) is a class of bacterial immune systems that degrade invading nucleic acids and prevent all modes of HGT (Marraffini, 2015). CRISPR loci consist of short sequences derived from past invaders, known as spacers, which are integrated between repeat sequences of similar length (~30-40 nucleotides). These repeat-spacer arrays encode small CRISPR RNAs (crRNAs) that associate with Cas proteins, forming a ribonucleoprotein complex that destroys foreign DNA and/or RNA in a sequence-dependent manner. Many staphylococci possess Type III-A CRISPR-Cas systems (Marraffini and Sontheimer, 2008; Golding et al., 2012; Cao et al., 2016). The Type III-A system in S. epidermidis RP62a, a wild-type human isolate (Christensen et al., 1987), encodes a multi-subunit complex called Cas10-Csm, composed of Cas10, Csm2, Csm3, Csm4, Csm5 and a crRNA (Hatoum-Aslan et al., 2013). This system has been shown to prevent the conjugative transfer of antibiotic resistance genes (Marraffini and Sontheimer, 2008; Hatoum-Aslan et al., 2014) and phage infection (Goldberg et al., 2014; Maniv et al., 2016), thus providing a natural barrier for HGT, and a model for Type III CRISPR-Cas systems in staphylococci.

The overexpression and purification of recombinant CRISPR-associated proteins from Escherichia coli (both the Cas10-Csm complex and individual subunits) followed by in vitro biochemical assays have revealed important insights into their functions (Hatoum-Aslan et al., 2013; Samai et al., 2015; Walker et al., 2016). However, such assays fail to 1) recover information about protein function and stability in the native cellular environment, and 2) identify biologically relevant binding partners that are not a part of the core Cas10-Csm complex. Indeed, purification of Cas10-Csm from its native S. epidermidis background has yielded additional insights into the genetic requirements for complex stability and function, crRNA processing, and non-Cas binding partners that might play a role in the CRISPR-Cas pathway (Hatoum-Aslan et al., 2013 and 2014; Walker et al., 2016). Here, we provide a detailed protocol for the purification of Cas10-Csm from S. epidermidis or S. aureus strains bearing the Type III-A CRISPR-Cas system on a plasmid. The protocol involves two affinity-purification steps that can be carried out over the course of five days (Figure 1). Importantly, this protocol can be easily adapted to study Cas10-Csm complexes in other Staphylococcus species, thus providing an essential tool to probe and understand these important immune systems.


Figure 1. Timeline of activities for expression and purification of the Cas10-Csm complex from staphylococci

Materials and Reagents

Note: Equivalent materials and reagents may be used as substitutes.

  1. Centrifuge tubes (50 ml) (VWR, catalog number: 21008-242 )
  2. Pipet tips with filter (0.1-10 µl) (VWR, catalog number: 89368-972 )
  3. Pipet tips with filter (1-200 µl) (VWR, catalog number: 89003-056 )
  4. Pipet tips with filter (100-1,000 µl) (VWR, catalog number: 89003-060 )
  5. PES membrane vacuum filter (0.22 µm) (VWR, catalog number: 10040-468 )
  6. Centrifuge tubes (15 ml) (VWR, catalog number: 21008-216 )
  7. Microcentrifuge tubes (VWR, catalog number: 87003-294 )
  8. Cellulose syringe filter (0.22 µm) (VWR, catalog number: 28145-477 )
  9. Petri dishes (100 x 15 mm) (VWR, catalog number: 25384-088 )
  10. Spectrophotometer cuvettes (VWR, catalog number: 97000-586 )
  11. Syringe (10 ml) (BD, catalog number: 309604 )
  12. Syringe (3 ml) (BD, catalog number: 309657 )
  13. Centrifugation polypropylene bottles (400 ml) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 75007585 )
  14. Disposable gravity flow columns for protein purification (Geno Technology, G-Biosciences, catalog number: 786-169 )
  15. S. epidermidis LM1680 expressing pcrispr/Csm26HN (Hatoum-Aslan et al., 2013) (see Note 1)
  16. S. aureus RN4220 expressing pcrispr/Csm26HN (Hatoum-Aslan et al., 2013) (see Note 1)
  17. HisPur Ni-NTA resin (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88222 )
  18. Sera-MagTM magnetic streptavidin coated beads (GE Healthcare, catalog number: 30152105011150 )
  19. SDS PAGE Gel 0.75 MM ‘Snap-A-GelsTM Mini Tris Glycine Precast Gels, Jule’ (VWR, catalog number: 66025-389 )
  20. Color protein standard ladder (New England Biolabs, catalog number: P7712S )
  21. BBLTM brain heart infusion (BHI) broth (BD, BBLTM, catalog number: 211060 )
  22. DifcoTM brain heart infusion (BHI) agar (BD, DifcoTM, catalog number: 241810 )
  23. Chloramphenicol (Alfa Aesar, catalog number: B20841 )
  24. 100% ethanol (Decon Labs, catalog number: V1016TP )
  25. Neomycin sulfate (AMRESCO, catalog number: 0558-25G )
  26. Magnesium chloride (MgCl2) (AMRESCO, catalog number: J364 )
  27. Tris (AMRESCO, catalog number: 0497 )
  28. Hydrochloric acid (HCl) (VWR, BDH®, catalog number: BDH3030-2.5LPC )
  29. Potassium chloride (KCl) (VWR, BDH®, catalog number: BDH9258-500G )
  30. EDTA, disodium salt, dihydrate (EMD Millipore, OmniPur®, catalog number: 4050 )
  31. Sodium hydroxide (NaOH) (AMRESCO, catalog number: 0583 )
  32. Sodium dodecyl sulfate (SDS) (VWR, catalog number: 97064-862 )
  33. Bromophenol blue (VWR, catalog number: 97061-690 )
  34. Ambicin® L (Recombinant lysostaphin) (AMBI, catalog number: LSPN-50 )
  35. Sodium acetate anhydrous (NaOAc) (AMRESCO, catalog number: 0602 )
  36. Sodium phosphate monobasic (NaH2PO4) (AMRESCO, catalog number: 0571 )
  37. Sodium chloride (NaCl) (VWR, BDH®, catalog number: BDH9286 )
  38. Glycerol (AMRESCO, catalog number: M152 )
  39. Beta-mercaptoethanol (β-ME) (Geno Technology, G-Biosciences, catalog number: BC98 )
  40. Coomassie Blue G-250 (AMRESCO, catalog number: M140-10G )
  41. Methanol (VWR, BDH®, catalog number: BDH20864.400 )
  42. Acetic acid (HAc) (VWR, BDH®, catalog number: BDH3096-2.5LPC )
  43. Tris-glycine-SDS 10x buffer (AMRESCO, catalog number: 0783-5L )
  44. Imidazole (Alfa Aesar, catalog number: A10221 )
  45. Pierce protease and phosphatase inhibitor mini tablets, EDTA-free (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88669 )
  46. Triton X-100 surfactant (EMD Millipore, catalog number: TX1568 )
  47. Potassium hydroxide (KOH) (Alfa Aesar, catalog number: 13451 )
  48. Media and antibiotics (see Recipes)
    1. Brain heart infusion (BHI) broth
    2. Chloramphenicol stock (10 mg/ml)
    3. Neomycin stock (15 mg/ml)
  49. Stock solutions (see Recipes)
    1. 1 M MgCl2
    2. 1 M Tris-HCl, pH 8.3
    3. 1 M Tris-HCl, pH 6.8
    4. 1 M KCl
    5. 500 mM EDTA, pH 8.0
    6. 10% sodium dodecyl sulfate (SDS)
    7. 1% bromophenol blue
    8. 2 mg/ml recombinant lysostaphin
    9. 2x lysis buffer
    10. 5x annealing buffer
    11. 5x protein loading buffer
    12. Coomassie Blue staining solution
    13. Destaining solution
    14. 1x Tris-glycine buffer
  50. Buffers to prepare on Day 4 for purification–Part 1 (see Recipes)
    1. Elution buffer
    2. Resuspension buffer
    3. Equilibration buffer
    4. Wash 1 buffer
    5. Wash 2 buffer

Equipment

  1. Eppendorf Research® plus pipettes set (Eppendorf, catalog number: 2231000222 ), or equivalent pipettes set with a range of 1 μl to 1,000 μl
  2. Media/storage bottles (250 ml) (VWR, catalog number: 10754-816 ), or equivalent
  3. Erlenmeyer flask (2 L) (VWR, catalog number: 10545-844 ), or equivalent
  4. Standard orbital shaker (VWR, model: 1000, catalog number: 89032-088 ), or equivalent shaker that can gently rotate for gel staining and destaining
  5. New Brunswick I26 incubating shakers (Eppendorf, New BrunswickTM, model: I26 , catalog number: M1324-0008), or equivalent shaking incubator that can maintain 37 °C and 180 rpm
  6. Heraeus Multifuge X1R centrifuge series (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM MultifugeTM X1R , catalog number: 75004251), or equivalent refrigerated centrifuge with a rotor capable of holding 400 ml bottles and applying 13,000 x g of centrifugal force
  7. General purpose water baths (VWR, model: VWR General Purpose Water Baths, catalog number: 89501-460 ), or equivalent capable of maintaining 37 °C
  8. Sonifier® S-450 Analog sonicator (Emerson, Branson, model: S-450 , catalog number: 101-063-198), or equivalent ultrasonic homogenizer with operating frequency of 20 kHz and tip diameter of 3.2 mm
  9. 30 ml beaker (Corning, PYREX®, catalog number: 1000-30 ) or equivalent
  10. Digital dry block heaters (VWR, catalog number: 12621-088 ), or equivalent block heater capable of heating up to 95 °C
  11. Magnetic bead separation rack (Thermo Scientific, catalog number: MR02 ), or equivalent rack capable of collecting magnetic beads
  12. Autoclave (Getinge), or equivalent autoclaving instrument capable of heating up to 121 °C while applying 15 atm pressure
  13. Graduated cylinder (100 ml) (VWR, catalog number: 65000-006 ), or equivalent
  14. Graduated cylinder (1 L) (VWR, catalog number: 65000-012 ), or equivalent
  15. Media/storage bottles (1 L) (VWR, catalog number: 10754-820 ), or equivalent
  16. Media/storage bottles (100 ml) (VWR, catalog number: 10754-814 ), or equivalent
  17. Media/storage bottles (500 ml) (VWR, catalog number: 10754-818 ), or equivalent
  18. UltrospecTM 10 cell density meter (GE Healthcare, catalog number: 80-2116-30 ), or equivalent spectrophotometer that can measure the density of cells in suspension at 600 nm
  19. Mini-PROTEAN tetra cell (Bio-Rad Laboratories, model: Mini-PROTEAN Tetra Cell, catalog number: 1658004EDU ), or equivalent vertical electrophoresis system
  20. pH meter AccumetTM AB15 plus basic (Fisher Scientific, model: Fisher ScientificTM accumetTM AB15+ Basic and BioBasicTM, catalog number: 13-636-AB15P ), or equivalent pH meter with a range of 0.000-10.000

Procedure

  1. Day 1. Preparation of media, reagents, and single colonies
    1. Prepare 100 ml of brain heart infusion (BHI) broth in a 250-ml bottle (for starting overnight cultures), and 1 L of BHI broth in a 2 L Erlenmeyer flask (for large-scale propagation and protein purification) (see Recipe 1).
    2. Prepare antibiotics listed in Recipe 1.
    3. Prepare all the stock solutions needed for the experiment (see Recipe 2).
    4. Streak out a freezer stock of S. epidermidis LM1680 bearing pcrispr/Csm26HN (or S. aureus RN4220 bearing pcrispr/Csm26HN) on a BHI agar plate containing 10 μg/ml chloramphenicol. If S. epidermidis is used, supplement media with 15 μg/ml neomycin (see Note 1).

  2. Day 2. Overnight culture preparation
    1. Transfer 10 ml of BHI broth into a 50-ml conical tube.
    2. Add 10 μl of 10 mg/ml chloramphenicol to select for pcrispr/Csm26HN. If S. epidermidis LM1680 is being used, add additionally 10 μl of 15 mg/ml neomycin. If other strains and plasmids are being used, supplement media with antibiotics as appropriate (see Note 1).
    3. With a sterile pipette tip, pick a single colony of S. epidermidis LM1680 bearing pcrispr/Csm26HN (or S. aureus RN4220 bearing pcrispr/Csm26HN).
    4. Re-suspend the colony into the 10 ml of autoclaved media containing appropriate antibiotic(s).
    5. Close loosely the 50-ml conical tube containing the autoclaved media, antibiotics, and colony of interest.
    6. Incubate the bacterial culture at 37 °C for 16-20 h in a shaking incubator set to 180 rpm.

  3. Day 3. Large-scale propagation of cells overexpressing the Cas10-Csm complex
    1. Into the 1 L of BHI prepared in the 2 L Erlenmeyer flask, add 1 ml of 10 mg/ml chloramphenicol. If S. epidermidis LM1680 is being used, add additionally 1 ml of 15 mg/ml neomycin.
    2. Inoculate the 1 L of BHI plus antibiotic(s) with the entire 10 ml of overnight culture prepared on Day 2.
      Note: The OD600 nm for overnight S. epidermidis and S. aureus cultures is approximately 5-6.
    3. Measure the initial OD600 nm of diluted culture with a cell density meter to facilitate estimation of time required to reach the final OD600 nm (see below).
    4. Incubate the bacterial culture at 37 °C in a shaking incubator set to 180 rpm.
    5. Let the bacterial culture grow until OD600 nm = 2.00 (approximately 7-9 h if starting with a fresh overnight culture and initial OD600 nm = 0.05).
    6. Distribute the 1 L of liquid culture into four 400-ml polypropylene bottles.
    7. Centrifuge cells at 5,000 x g, 4 °C for 10 min.
    8. Discard supernatant and resuspend each pellet on ice with 10 ml of ice-cold distilled H2O (dH2O).
    9. Combine cell suspensions from all pellets into a clean 50 ml conical tube (on ice) and centrifuge at 5,000 x g, at 4 °C for 10 min.
    10. Discard supernatant and store pelleted cells in -80 °C (for optimal purification up to a week later) or proceed directly to purification.

  4. Day 4. Purification–Part 1
    1. Thaw the cell pellet on ice for 1 h.
    2. Prepare all buffers described in Recipe 3.
    3. Resuspend thawed pellet in 9 ml of ice-cold dH2O.
    4. Add 200 µl of 1 M MgCl2 and 125 µl of freshly thawed lysostaphin (2 mg/ml stock).
    5. Incubate the cell suspension for 1 h in a 37 °C water bath. Invert the tube several times after 30 min of incubation to homogenize the suspension.
    6. Add 10 ml of resuspension buffer to the lysed cells and invert tube several times. The cell lysate will become very viscous (see Figure 2).


      Figure 2. Viscosity of cell lysate before and after sonication of S. epidermidis LM1680 cells expressing pcrispr/Csm26HN. Before sonication (left), the cell lysate is milky white with the consistency of mucus. After sonication (right), the cell lysate becomes tinted brown with a more watery consistency.

    Important Note: All subsequent steps should be performed on ice or at 4 °C.
    1. Adjust the Sonifier S-450 to power level 6.0 and duty cycle percent to 6.
      Note: These settings correspond to a 60 W power output with a fixed repetition rate of 1 pulse per second. A duty cycle of ‘6’ indicates that power will be delivered 60% of each 1 sec pulse (with a rest 40% of each second).
    2. Pour the lysate into a 30-ml beaker set in an ice bucket. The cell lysate should occupy ~⅔ of the beaker’s volume such that lysate within has at least 1 inch depth.
    3. Immerse the sonicator tip into the lysate at a depth of ~½-⅔ inch such that there remains ~½ an inch of clearance from the bottom of the beaker.
      Important Note: The tip should not touch the sides or bottom of the beaker.
    4. Sonicate the lysate on ice for 30 sec at a time with one minute rest period in between. This cycle should be repeated three times or until the lysate has a more watery consistency. Often, the lysate will also acquire a brown tint (see Figure 2).
    5. Pour the lysate back into a 50-ml conical tube and centrifuge for 20 min, 4 °C, at 10,000 x g. The clarified lysate after centrifugation often retains the brownish tinge (see Figure 3).


      Figure 3. Appearance of sonicated cell lysate following the first centrifugation. After the cell debris is pelleted, the clarified lysate retains a yellow-brown tinge.

    6. Decant the supernatant into a fresh 50-ml conical tube and centrifuge for 20 min, 4 °C, at 13,000 x g.
      Note: The cell pellet may be saved and checked later on SDS-PAGE for the presence of the Cas10-Csm complex to assess its solubility.
    7. During the two spins, prepare/pack the column (to be done in the cold room or inside a deli refrigerator):
      1. Pipet 1 ml of slurry Ni-NTA resin into a disposable gravity flow column and allow the liquid to flow through.
      2. Equilibrate the Ni-NTA resin by passing 10 ml of equilibration buffer through the column at 4 °C.
      3. Cap the column once most of the buffer has passed through and ~2-3 mm of buffer remains atop the resin.
    8. After the final spin (step D12), pass the lysate through a 0.22 µm PES membrane vacuum filter and catch the filtrate into a sterile bottle.
    9. Pass the filtered lysate through the packed Ni-NTA column (prepared in step D13) and collect the flow through in a 50-ml conical tube labeled ‘lysate flow-through’.
      Note: The lysate flow-through can be checked later on SDS-PAGE for the presence of the Cas10-Csm complex in case the complex was suspected to have passed through the column without binding.
    10. Wash the column by passing 10 ml of wash 1 buffer through the column.
    11. Collect wash 1 flow-through into a 15-ml conical tube.
      Note: The wash 1 flow-through may be saved and checked later for the Cas10-Csm complex in case the complex was suspected to have passed through the column during the wash.
    12. Wash the column a second time by passing 10 ml of wash 2 buffer through the column.
    13. Collect wash 2 flow-through into a 15-ml conical tube.
      Note: The wash 2 flow-through may be saved and checked later for the Cas10-Csm complex in case the complex was suspected to have passed through the column during the wash.
    14. Elute proteins from the column with 5-7 aliquots of elution buffer (500 µl for each fraction). As each aliquot flows through, capture it into a fresh, appropriately-labeled microcentrifuge tube.
    15. Into fresh labeled microcentrifuge tubes, combine 20 µl of each fraction with 5 µl of 5x protein loading buffer.
    16. Heat samples at 95 °C for 5 min on a heating block.
    17. Load samples alongside a protein standard into a 12% SDS-PAGE gel.
    18. Run the gel in Tris-glycine-SDS 1x buffer at 120 V for 1 h.
    19. Stain the gel with Coomassie Blue solution for 10 min and destain with destaining solution for up to 40 min.
    20. Image the gel under white light (Figure 4).
    21. Save the desired elutions at -20 °C (see Note 2).
      Note: Avoid repeated freeze-thaw cycles of the complex as this leads to protein aggregation. If necessary, aliquot the desired elutions into smaller volumes (~100 µl). Protein complexes that have not been repeatedly thawed can be stored in the elution buffer up to a year at -20 °C without noticeable degradation of protein or crRNAs. 


      Figure 4. Elutions of the Cas10-Csm complex following purification from S. epidermidis LM1680 expressing pcrispr/Csm26HN. Shown are the seven fractions (E1-E7) collected from a nickel-agarose column and analyzed on a 12% SDS PAGE gel. Proteins were stained with Coomassie Blue G-250.

  5. Day 5. Purification–Part 2 
    1. Pipette 10 µl of Sera-MagTM magnetic streptavidin coated beads into a microcentrifuge tube.
      Note: Mix the bead suspension well by pipetting up and down several times before use.
    2. Resuspend the beads with 100 µl of 1x annealing buffer (see Recipe 2).
      Note: Bead re-suspension is achieved once the solution becomes a homogenous brown color and no beads accumulation is seen at the side or bottom of the tube. When tubes are removed from the magnetic rack, the beads within can be easily resuspended.
    3. Pellet the beads by placing the microcentrifuge tube into a magnetic separation rack and waiting for the beads to collect on the side of the tube.
      Note: The collection process can vary from 30 sec up to 2 min depending on beads and rack used. The collection process will be completed once the supernatant becomes clear and the beads (brown color) aggregate on the side of the tube where the magnet is located. If the beads are broadly spread around the side of the tube, twist the microcentrifuge tube a few degrees clockwise, or counter-clockwise within the rack to pull all beads closer together.
    4. Gently pipette out the supernatant without disturbing the beads.
    5. Repeat the wash (steps E2-E4) three times.
    6. Pool the most concentrated Cas10-Csm complex fractions (obtained in Day 4) and combine with 2 ng of a 5’-biotinylated oligonucleotide antisense to a crRNA (see Note 3).
    7. Let the mixture incubate for 30 min at room temperature.
    8. Remove the microcentrifuge tube that contains the beads away from the magnetic rack and add the Cas10-Csm complex/oligo mixture into the equilibrated streptavidin beads.
    9. Resuspend beads until the mixture appears homogenous and let it anneal at room temperature for 30 min.
    10. Collect beads to the side of the tube by placing the microcentrifuge tube back into the magnetic rack ~30 sec.
    11. Gently pipette out the supernatant without disturbing the beads.
    12. Remove tube from the magnetic rack and resuspend the beads in 100 µl of 1x annealing buffer.
    13. Collect beads to the side of the tube by placing the microcentrifuge tube back into the magnetic rack ~30 sec.
    14. Gently pipette out the supernatant without disturbing the beads.
    15. Repeat the wash (steps E12-E14) two more times.
    16. Add 20 µl of 1x annealing buffer to the beads and 5 μl of 5x protein loading buffer.
    17. Heat the samples for 5 min at 95 °C on a heating block.
    18. Place the tube back into the magnetic rack to remove the beads from solution.
    19. Load the supernatant alongside a protein standard into a 12% SDS-PAGE gel.
    20. Run the gel in Tris-glycine-SDS 1x buffer at 120 V for 1 h.
    21. Stain the gel with Coomassie Blue solution for 10 min and destain with destaining solution for up to 40 min.
    22. Image the gel under white light (Figure 5).


      Figure 5. S. epidermidis Cas10-Csm complex collected from magnetic streptavidin beads following an affinity purification with a biotinylated oligonucleotide antisense to spc1 crRNAs. Proteins were resolved on a 12% SDS PAGE gel and visualized with Coomassie Blue G250.

Data analysis

This assay is used as a qualitative measure to assess Cas10-Csm complex integrity. The presence or absence of a particular protein subunit on the SDS-PAGE gel will indicate whether or not it is stably associated with the complex. Using this protocol, the presence or absence and lengths of crRNAs within the complex can be determined by extracting crRNAs directly from Cas10-Csm complex aliquots obtained from the 1st purification process, followed by 5’-end labeling with γ-32P-ATP, and resolving these on an 8-12% polyacrylamide gel. Before a definitive determination of complex stability can be made, these assays must give consistent results over three independent replicates. For complexes that are being characterized for the first time, the identity of each subunit in the gel must be confirmed using Western blotting. These additional methods are described in (Hatoum-Aslan et al., 2013).

Notes

  1. This protocol describes the purification of Cas10-Csm complexes from S. epidermidis LM1680 or S. aureus RN4220 cells bearing pcrispr/Csm26HN, a plasmid that encodes the entire Type III-A CRISPR-Cas system from S. epidermidis RP62a with a 6x-His tag introduced into the N-terminus of csm2 (Hatoum-Aslan et al., 2013). S. epidermidis LM1680 is a derivative of S. epidermidis RP62a with a chromosomal deletion encompassing the CRISPR-Cas locus (Hatoum-Aslan et al., 2013). S. aureus RN4220 is a crispr-strain (Nair et al., 2011) that is used as a cloning intermediate. When re-introduced on pcrispr/Csm2H6N, the CRISPR-cas system retains full functionality in both strains. pcrispr/Csm2H6N requires selection in 10 µg/ml of chloramphenicol. Additionally, 15 µg/ml of neomycin is used to select specifically for S. epidermidis RP62a or LM1680. This protocol can also be adapted to overexpress the CRISPR-Cas system on different plasmids, or in different strains, however, antibiotic selection would also have to be modified accordingly. If working with wild-type strains, overexpressing a plasmid encoding the CRISPR-Cas system with one subunit 6x-His tagged should, in theory, allow for Cas10-Csm complex purification even in the presence of the chromosomal copy of the system in the background.
  2. Mass spectrometry analysis may be performed following the first purification to detect potential binding partners loosely associated with the complex. In addition, the presence or absence of crRNAs within complexes and their lengths can be determined by extracting RNAs from complexes following the first purification, end-labeling the RNAs, and resolving on a urea PAGE gel. If a cleaner prep is desired or to assess complex stability, the second purification described for day 5 should be performed.
  3. The sequence of the biotinylated oligonucleotide used in this example is complementary to the first spacer (spc1) of the repeat-spacer array:
    5’-Biotin-TEG-ACGAGAACACGUAUGCCGAAGUAUAUAAAUC

Recipes

  1. Media and antibiotics
    1. Brain heart infusion (BHI) broth
      1. Dissolve 3.7 g per 100 ml dH2O
      2. Autoclave at 121 °C for 30 min
      3. Store at room temperature
    2. Chloramphenicol stock (10 mg/ml)
      1. Dissolve 100 mg chloramphenicol into 10 ml of 100% ethanol
      2. Pass through a 0.22 μm syringe filter
      3. Store at 4 °C
    3. Neomycin stock (15 mg/ml)
      1. Dissolve 150 mg neomycin into 10 ml dH2O
      2. Pass through a 0.22 μm syringe filter
      3. Store at 4 °C
  2. Stock solutions
    1. 1 M MgCl2
      1. Place 9.521 g MgCl2 into a 100-ml graduated cylinder
      2. Fill with dH2O up to 100 ml
      3. Store in a bottle at room temperature
    2. 1 M Tris-HCl, pH 8.3
      1. Place 121.14 g Tris into a 1-L graduated cylinder
      2. Dissolve with 800 ml of dH2O
      3. Adjust pH to 8.3 with 12 N HCl
      4. Fill with dH2O up to 1 L
      5. Store in a bottle at room temperature
    3. 1 M Tris-HCl, pH 6.8
      1. Place 121.14 g Tris into a 1-L graduated cylinder
      2. Dissolve with 800 ml of dH2O
      3. Adjust pH to 6.8 with 12 N HCl
      4. Fill with dH2O up to 1 L
      5. Store in a bottle at room temperature
    4. 1 M KCl
      1. Place 7.455 g KCl into a 100-ml graduated cylinder
      2. Fill with dH2O up to 100 ml
      3. Store in a bottle at room temperature
    5. 500 mM EDTA, pH 8.0
      1. Place 93.06 g EDTA, disodium salt, dihydrate (MW = 372.24 g/mol) into a 500-ml beaker
      2. Dissolve as much as possible with 350 ml of dH2O
      3. Adjust pH to 8.0 with 10 N NaOH while constantly stirring
        Note: The EDTA will not dissolve completely unless its pH is at 8.0. However, the pH of EDTA continually drops as it gets dissolved. Therefore, constant addition of 10 N NaOH must be used to get the EDTA completely dissolved with a final pH of 8.0.
      4. Fill with dH2O up to 500 ml
      5. Store in a bottle at room temperature
    6. 10% sodium dodecyl sulfate (SDS)
      1. Place 1 g of SDS into a 15-ml conical tube
      2. Dissolve with 6 ml of dH2O
      3. Fill with dH2O up to 10 ml
      4. Store at room temperature

        Note: Since powdered SDS can cause serious eye and lung damage, a 20% solution may be purchased (VWR Cat. No. 97062-440) and diluted with dH2O (1:1).

    7. 1% bromophenol blue
      1. Place 0.1 g of bromophenol blue into a 15-ml conical tube
      2. Fill with dH2O up to 10 ml
      3. Store at room temperature
    8. 2 mg/ml recombinant lysostaphin
      1. Dissolve 50 mg lysostaphin in 25 ml NaOAc, pH 4.5
      2. Distribute 0.5 ml aliquots into microcentrifuge tubes
      3. Store at -80 °C
      4. Thaw just prior to use
    9. 2x lysis buffer
      1. Place 11.998 g NaH2PO4 into a 1-L graduated cylinder ([final] = 100 mM)
      2. Place 35.064 g NaCl into the graduated cylinder ([final] = 600 mM)
      3. Fill to 800 ml with dH2O
      4. Adjust pH to 8.0 with 10 N NaOH
      5. Fill up to 1 L
      6. Store at room temperature
    10. 5x annealing buffer
      1. Place 2.5 ml of 1 M Tris-HCl, pH 8.3 into a 100-ml bottle ([final] = 25 mM)
      2. Place 37.5 ml of 1 M KCl into the bottle ([final] = 375 mM)
      3. Place 1 ml of 500 mM EDTA, pH 8.0 into the bottle ([final] = 5 mM)
      4. Add 59 ml of dH2O
      5. Store bottle at room temperature
    11. 5x protein loading buffer
      1. Place 0.5 ml of 1 M Tris-HCl, pH 6.8 into a 15-ml conical tube ([final] = 62.5 mM)
      2. Place 0.8 ml of 100% glycerol into the tube ([final] = 10%)
      3. Place 1.6 ml of 10% SDS solution into the tube ([final] = 2%)
      4. Place 0.4 ml of 14.3 M β-mercaptoethanol ([final] = 715 mM)
      5. Add 0.4 ml of 1% bromophenol blue into the tube ([final] = 0.05%)
      6. Add 4.3 ml of dH2O
      7. Distribute 1 ml aliquots into microcentrifuge tubes
      8. Store at -20 °C
    12. Coomassie Blue staining solution 
      1. Place 1 g of Coomassie Blue G-250 into a 1-L graduated cylinder ([final] = 0.1%)
      2. Place 500 ml of methanol into the graduated cylinder ([final] = 50%)
      3. Place 100 ml of acetic acid into the graduated cylinder ([final] = 10%)
      4. Place 400 ml of dH2O into the graduated cylinder ([final] = 40%)
      5. Store in a bottle at room temperature
    13. Destaining solution
      1. Place 500 ml of methanol into a 1-L graduated cylinder ([final] = 50%)
      2. Place 100 ml of acetic acid into the graduated cylinder ([final] = 10%)
      3. Place 400 ml of dH2O into the graduated cylinder ([final] = 40%)
      4. Store in a bottle at room temperature
    14. 1x Tris-glycine-SDS buffer
      1. Place 100 ml of 10x Tris-glycine-SDS buffer into a 1-L graduated cylinder
      2. Fill with dH2O up to 1 L
      3. Store in a bottle at room temperature
  3. Buffers to prepare on Day 4 for purification–Part 1
    Note: All the following buffers should be freshly prepared. Keep buffers on ice. Prepare 10 ml of each buffer per cell pellet from 1 L of culture.
    1. Elution buffer (10 ml [Final volume])
      1. Place 60 mg NaH2PO4 into a 50-ml conical tube ([final] = 50 mM)
      2. Add 175 mg NaCl ([final] = 300 mM)
      3. Add 170 mg imidazole ([final] = 250 mM)
      4. Add 1 ml glycerol ([final] = 10%)
      5. Add 8 ml dH2O
      6. Adjust pH to 8.0 with 10 N NaOH
      7. Bring final volume up to 10 ml with dH2O
    2. Resuspension buffer (10 ml [Final volume])
      1. Place 9.2 ml 2x lysis buffer into a 50-ml conical tube
      2. Add 800 µl elution buffer
      3. Add 1 cOmplete tablet protease inhibitor (free EDTA)
      4. Cap tube tightly and vortex until the tablet has completely dissolved (30 sec-1 min)
      5. Add 10 µl Triton X-100 ([final] = 0.1%)
      6. Invert tube gently until the Triton X-100 has completely dissolved
    3. Equilibration buffer (10 ml [Final volume])
      1. Place 5 ml 2x lysis buffer into a 50-ml conical tube
      2. Add 5 ml dH2O
    4. Wash 1 buffer (10 ml [Final volume])
      1. Place 5 ml 2x lysis buffer into a 50-ml conical tube
      2. Add 800 µl elution buffer
      3. Add 4.2 ml dH2O
    5. Wash 2 buffer ( 10 ml [Final volume])
      1. Place 5 ml 2x lysis buffer into a 50-ml conical tube
      2. Add 800 µl elution buffer
      3. Add 3.2 ml dH2O
      4. Add 1 ml glycerol ([final] = 10%)

Acknowledgments

A. H-A. is supported by the University of Alabama (UA) College of Arts and Sciences; a grant from the UA College Academy of Research, Scholarship, and Creative Activity (CARSCA); and the National Institutes of Health [5K22AI113106-02]. This protocol was adapted from that published in Hatoum-Aslan et al., J Biol Chem, 2013.

References

  1. Cao, L., Gao, C. H., Zhu, J., Zhao, L., Wu, Q., Li, M. and Sun, B. (2016). Identification and functional study of type III-A CRISPR-Cas systems in clinical isolates of Staphylococcus aureus. Int J Med Microbiol 306(8): 686-696.
  2. Christensen, G. D., Baddour, L. M. and Simpson, W. A. (1987). Phenotypic variation of Staphylococcus epidermidis slime production in vitro and in vivo. Infect Immun 55(12): 2870-2877.
  3. Cogen, A. L., Yamasaki, K., Sanchez, K. M., Dorschner, R. A., Lai, Y., MacLeod, D. T., Torpey, J. W., Otto, M., Nizet, V., Kim, J. E. and Gallo, R. L. (2010). Selective antimicrobial action is provided by phenol-soluble modulins derived from Staphylococcus epidermidis, a normal resident of the skin. J Invest Dermatol 130(1): 192-200.
  4. Conlan, S., Kong, H. H. and Segre, J. A. (2012). Species-level analysis of DNA sequence data from the NIH Human Microbiome Project. PLoS One 7(10): e47075.
  5. Furuya, E. Y. and Lowy, F. D. (2006). Antimicrobial-resistant bacteria in the community setting. Nat Rev Microbiol 4(1): 36-45.
  6. Goldberg, G. W., Jiang, W., Bikard, D. and Marraffini, L. A. (2014). Conditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting. Nature 514(7524): 633-637.
  7. Golding, G. R., Bryden, L., Levett, P. N., McDonald, R. R., Wong, A., Graham, M. R., Tyler, S., Van Domselaar, G., Mabon, P., Kent, H., Butaye, P., Smith, T. C., Kadlec, K., Schwarz, S., Weese, S. J. and Mulvey, M. R. (2012). Whole-genome sequence of livestock-associated st398 methicillin-resistant staphylococcus aureus Isolated from Humans in Canada. J Bacteriol 194(23): 6627-6628.
  8. Grice, E. A. and Segre, J. A. (2011). The skin microbiome. Nat Rev Microbiol 9(4): 244-253.
  9. Harris, L. G. and Richards, R. G. (2006). Staphylococci and implant surfaces: a review. Injury 37 Suppl 2: S3-14.
  10. Hatoum-Aslan, A., Maniv, I., Samai, P. and Marraffini, L. A. (2014). Genetic characterization of antiplasmid immunity through a type III-A CRISPR-Cas system. J Bacteriol 196(2): 310-317.
  11. Hatoum-Aslan, A., Samai, P., Maniv, I., Jiang, W. and Marraffini, L. A. (2013). A ruler protein in a complex for antiviral defense determines the length of small interfering CRISPR RNAs. J Biol Chem 288(39): 27888-27897.
  12. Iwase, T., Uehara, Y., Shinji, H., Tajima, A., Seo, H., Takada, K., Agata, T. and Mizunoe, Y. (2010). Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonization. Nature 465(7296): 346-349.
  13. Lai, Y., Cogen, A. L., Radek, K. A., Park, H. J., Macleod, D. T., Leichtle, A., Ryan, A. F., Di Nardo, A. and Gallo, R. L. (2010). Activation of TLR2 by a small molecule produced by Staphylococcus epidermidis increases antimicrobial defense against bacterial skin infections. J Invest Dermatol 130(9): 2211-2221.
  14. Lowy, F. D. (1998). Staphylococcus aureus infections. N Engl J Med 339(8): 520-532.
  15. Maniv, I., Jiang, W., Bikard, D. and Marraffini, L. A. (2016). Impact of different target sequences on type III CRISPR-Cas immunity. J Bacteriol 198(6): 941-950.
  16. Marraffini, L. A. (2015). CRISPR-Cas immunity in prokaryotes. Nature 526(7571): 55-61.
  17. Marraffini, L. A. and Sontheimer, E. J. (2008). CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322(5909): 1843-1845.
  18. Naik, S., Bouladoux, N., Linehan, J. L., Han, S. J., Harrison, O. J., Wilhelm, C., Conlan, S., Himmelfarb, S., Byrd, A. L., Deming, C., Quinones, M., Brenchley, J. M., Kong, H. H., Tussiwand, R., Murphy, K. M., Merad, M., Segre, J. A. and Belkaid, Y. (2015). Commensal-dendritic-cell interaction specifies a unique protective skin immune signature. Nature 520(7545):104-108.
  19. Nair, D., Memmi, G., Hernandez, D., Bard, J., Beaume, M., Gill, S., Francois, P., Cheung, A. L. (2011). Whole-genome sequencing of Staphylococcus aureus strain RN4220, a key laboratory strain used in virulence research, identifies mutations that affect not only virulence factors but also the fitness of the strain. J Bacteriol 193(9): 2332-2335.
  20. National Nosocomial Infections Surveillance, S. (2004). National Nosocomial Infections Surveillance (NNIS) System Report, data summary from January 1992 through June 2004, issued October 2004. Am J Infect Control 32(8): 470-485.
  21. Otto, M. (2009). Staphylococcus epidermidis – the “accidental” pathogen. Nat Rev Microbiol 7(8):555-567.
  22. Samai, P., Pyenson, N., Jiang, W., Goldberg, G. W., Hatoum-Aslan, A. and Marraffini, L. A. (2015). Co-transcriptional DNA and RNA cleavage during type III CRISPR-Cas immunity. Cell 161(5): 1164-1174.
  23. Stryjewski, M. E. and Chambers, H. F. (2008). Skin and soft-tissue infections caused by community-acquired methicillin-resistant Staphylococcus aureus. Clin Infect Dis 46 Suppl 5: S368-377.
  24. Walker, F. C., Chou-Zheng, L., Dunkle, J. A. and Hatoum-Aslan, A. (2016). Molecular determinants for CRISPR RNA maturation in the Cas10-Csm complex and roles for non-Cas nucleases. Nucleic Acids Res. 45(4): 2112-2123.

简介

CRISPR-Cas(聚集的定期间隔的短回文重复-CRISPR相关蛋白)是一类以序列特异性方式降解外来核酸的原核免疫系统。这些系统依赖于由Cas核酸酶和小型CRISPR RNA(crRNA)组成的核糖核蛋白复合物。表皮葡萄球菌和金黄色葡萄球菌是人皮肤上的细菌居民,也是抗生素抗性感染的主要原因(Lowy,1998; National Nosocomial Infection Surveillance,2004; Otto,2009) 。许多葡萄球菌具有III型A CRISPR-Cas系统(Marraffini和Sontheimer,2008; Cao等人,2016),已被证明可预防质粒转移并防止病毒性捕食者(Goldberg这些生物体中,等等,2014; Hatoum-Aslan等人,2014; Samai等人,2015)。因此,在天然葡萄球菌背景中获得对这些系统的机械理解可以导致对影响这些病原体的进化和存活的因素的重要见解。 III-A型CRISPR-Cas系统编码称为Cas10-Csm的五亚单位效应复合物(Hatoum-Aslan等人,2013)。在这里,我们描述了一种用于表达和纯化Cas10-Csm的方法。表皮样背景或异源性S。金黄色葡萄球菌背景。该方法由两步纯化方案组成,包括Ni 2 + - 亲和层析和DNA亲和生物素下拉,它们共同产生了Cas10-Csm复合物的纯制剂。以前已经使用这种方法来分析突变对Cas10-Csm复合物完整性的影响(Hatoum-Aslan等人,2014),crRNA形成(Hatoum-Aslan等人, ,2013),并检测与核心Cas10-Csm复合物直接相互作用的绑定伙伴(Walker等人,2016)。重要的是,这种方法可以很容易地适用于其他<葡萄球菌>物种,以探测和了解其本土的III-A型CRISPR-Cas系统。

背景 表皮葡萄球菌和金黄色葡萄球菌是具有一定范围的相反影响的普遍的皮肤细菌。虽然,金黄色葡萄球菌无症状地占据了约30%的人群(Conlan等人,2012),这种生物体是皮肤和软组织感染的主要原因(Stryjewski和Chambers,2008; Grice和Segre,2011)。相比之下,表皮炎通常被认为是有益的,并且通过以下方式促进人体健康:1)防止S。金霉素定植(Iwase等人,2010),2)产生靶向皮肤病原体的抗微生物肽(Cogen等人,2010),以及3)刺激人类免疫系统以促进病原体的防御(Lai等人,2010; Naik等人,2015)。然而,当允许其破坏皮肤屏障时,该物种也可引起抗生素抗性感染,特别是在留置医疗器械上(Otto,2009; Harris和Richards,2006)。此外,最近在医院和社区环境中出现了抗所有已知抗生素的致病性葡萄球菌(Furuya和Lowy,2006),并已成为全球公共卫生的主要威胁。水平基因转移(HGT)或相关细菌物种之间遗传信息的交换是这些生物获得毒力因子和多重耐药性的主要途径。因此,了解在这些生物体内影响和调节HGT的因素至关重要。
&NBSP; CRISPR-Cas(集群定期间隔的短回文重复CRISPR相关蛋白)是一类细菌免疫系统,可降解入侵核酸并阻止所有模式的HGT(Marraffini,2015)。 CRISPR基因座由来自过去侵入者的短序列组成,称为间隔区,它们被整合在相似长度(〜30-40个核苷酸)的重复序列之间。这些重复间隔阵列编码与Cas蛋白相关的小型CRISPR RNA(crRNA),形成以序列依赖的方式破坏外来DNA和/或RNA的核糖核蛋白复合物。许多葡萄球菌具有III-A型CRISPR-Cas系统(Marraffini和Sontheimer,2008; Golding等人,2012; Cao等人,2016)。 III型A系统。表皮葡萄球菌RP62a是野生型人类分离物(Christensen等人,1987),编码称为Cas10-Csm的多亚基复合物,其由Cas10,Csm2,Csm3,Csm4, Csm5和crRNA(Hatoum-Aslan等人,2013)。已经证明该系统可以防止抗生素抗性基因的共转移(Marraffini和Sontheimer,2008; Hatoum-Aslan等人,2014)和噬菌体感染(Goldberg等人,2014年; Maniv等人,2016),从而为HGT提供了天然的屏障,也是葡萄球菌中III型CRISPR-Cas系统的模型。
&NBSP;来自大肠杆菌(Cas10-Csm复合物和单个亚基)的重组CRISPR相关蛋白的过表达和纯化随后在体外生物化学测定中已经揭示了重要的见解他们的功能(Hatoum-Aslan等人,2013; Samai等人,2015; Walker等人,2016)。然而,这种测定不能1)恢复关于天然细胞环境中蛋白质功能和稳定性的信息,以及2)识别不是核心Cas10-Csm复合物的一部分的生物相关结合配偶体。事实上,Cas10-Csm从其原生质体纯化。表皮葡萄球菌背景已经对复杂的稳定性和功能,crRNA加工和可能在CRISPR-Cas途径中起作用的非Cas结合配偶体的遗传要求进行了进一步的了解(Hatoum-Aslan等,2013和2014; Walker等人,2016)。在这里,我们提供了一个详细的解决方案,用于从S&S提纯Cas10-Csm。表皮细胞或 S。在质粒上携带III-A型CRISPR-Cas系统的金黄色葡萄球菌菌株。该方案涉及两个亲和纯化步骤,可以在五天的时间内进行(图1)。重要的是,该方案可以很容易地适应于研究其他葡萄球菌物种中的Cas10-Csm复合物,从而提供了探索和了解这些重要免疫系统的重要工具。


图1.来自葡萄球菌的Cas10-Csm复合物的表达和纯化的活性时间表

关键字:CRISPR-Cas Type III-A, Cas10-Csm,  葡萄球菌,  蛋白质纯化,  蛋白质表达,  DNA亲和层析,  生物素 pull-down

材料和试剂

注意:等效的材料和试剂可用作替代品。

  1. 离心管(50ml)(VWR,目录号:21008-242)
  2. 带过滤器的吸头(0.1-10μl)(VWR,目录号:89368-972)
  3. 带过滤器的吸头(1-200μl)(VWR,目录号:89003-056)
  4. 带过滤器的吸头(100-1,000μl)(VWR,目录号:89003-060)
  5. PES膜真空过滤器(0.22μm)(VWR,目录号:10040-468)
  6. 离心管(15 ml)(VWR,目录号:21008-216)
  7. 微量离心管(VWR,目录号:87003-294)
  8. 纤维素注射器过滤器(0.22μm)(VWR,目录号:28145-477)
  9. 培养皿(100 x 15毫米)(VWR,目录号:25384-088)
  10. 分光光度计比色皿(VWR,目录号:97000-586)
  11. 注射器(10ml)(BD,目录号:309604)
  12. 注射器(3 ml)(BD,目录号:309657)
  13. 离心聚丙烯瓶(400ml)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:75007585)
  14. 用于蛋白质纯化的一次性重力流动柱(Geno Technology,G-Biosciences,目录号:786-169)
  15. S上。表达表达 LM1680表达p crispr / Csm2 <6h> (Hatoum-Aslan等人,2013)(参见注1) />
  16. S上。金黄色葡萄球菌 RN4220表达p / Csm2 <6h> (Hatoum-Aslan等人,2013)(参见注1) />
  17. HisPur Ni-NTA树脂(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:88222)
  18. Sera-Mag TM 磁性链亲和素包被的珠粒(GE Healthcare,目录号:30152105011150)
  19. SDS PAGE Gel 0.75 MM'Snap-A-Gels TM Mini Tris Glycine Precast Gels,Jule'(VWR,目录号:66025-389)
  20. 彩色蛋白标准梯(New England Biolabs,目录号:P7712S)
  21. 脑内输注(BHI)肉汤(BD,BBL TM,目录号:211060)的BBL TM
  22. Difco TM脑输注(BHI)琼脂(BD,Difco TM,目录号:241810)
  23. 氯霉素(Alfa Aesar,目录号:B20841)
  24. 100%乙醇(Decon Labs,目录号:V1016TP)
  25. 硫酸新霉素(AMRESCO,目录号:0558-25G)
  26. 氯化镁(MgCl 2)(AMRESCO,目录号:J364)
  27. Tris(AMRESCO,目录号:0497)
  28. 盐酸(HCl)(VWR,BDH ,目录号:BDH3030-2.5LPC)
  29. 氯化钾(KCl)(VWR,BDH ,目录号:BDH9258-500G)
  30. EDTA,二钠盐,二水合物(EMD Millipore,OmniPur,目录号:4050)
  31. 氢氧化钠(NaOH)(AMRESCO,目录号:0583)
  32. 十二烷基硫酸钠(SDS)(VWR,目录号:97064-862)
  33. 溴酚蓝(VWR,目录号:97061-690)
  34. Ambicin ®(重组溶葡萄球菌素)(AMBI,目录号:LSPN-50)
  35. 无水乙酸钠(NaOAc)(AMRESCO,目录号:0602)
  36. 磷酸二氢钠(NaH 2 PO 4)(AMRESCO,目录号:0571)
  37. 氯化钠(NaCl)(VWR,BDH ,目录号:BDH9286)
  38. 甘油(AMRESCO,目录号:M152)
  39. β-巯基乙醇(β-ME)(Geno Technology,G-Biosciences,目录号:BC98)
  40. 考马斯蓝G-250(AMRESCO,目录号:M140-10G)
  41. 甲醇(VWR,BDH ®,目录号:BDH20864.400)
  42. 乙酸(HAc)(VWR,BDH ,目录号:BDH3096-2.5LPC)
  43. Tris-glycine-SDS 10x缓冲液(AMRESCO,目录号:0783-5L)
  44. 咪唑(Alfa Aesar,目录号:A10221)
  45. Pierce蛋白酶和磷酸酶抑制剂迷你片,不含EDTA(Thermo Fisher Scientific,Thermo Scientific TM,目录号:88669)
  46. Triton X-100表面活性剂(EMD Millipore,目录号:TX1568)
  47. 氢氧化钾(KOH)(Alfa Aesar,目录号:13451)
  48. 媒体和抗生素(见配方)
    1. 脑心脏输液(BHI)肉汤
    2. 氯霉素原料(10mg / ml)
    3. 新霉素原料(15mg / ml)
  49. 库存解决方案(见配方)
    1. 1 M MgCl 2
    2. 1M Tris-HCl,pH 8.3
    3. 1M Tris-HCl,pH6.8。
    4. 1 M KCl
    5. 500mM EDTA,pH 8.0
    6. 10%十二烷基硫酸钠(SDS)
    7. 1%溴酚蓝
    8. 2 mg / ml重组溶葡萄球菌素
    9. 2x裂解缓冲液
    10. 5x退火缓冲液
    11. 5x蛋白加载缓冲液
    12. 考马斯蓝染色溶液
    13. 解决方案
    14. 1x Tris-甘氨酸缓冲液
  50. 缓冲液在第4天准备净化 - 第1部分(见配方)
    1. 洗脱缓冲液
    2. 再悬浮缓冲液
    3. 平衡缓冲器
    4. 洗1个缓冲区
    5. 洗2缓冲液

设备

  1. Eppendorf Research ®加移液器组(Eppendorf,目录号:2231000222)或等效移液管,设定范围为1μl至1,000μl
  2. 介质/储存瓶(250毫升)(VWR,目录号:10754-816)或等效物
  3. 锥形瓶(2升)(VWR,目录号:10545-844)或等效物
  4. 标准轨道摇床(VWR,型号:1000,目录号:89032-088),或可以轻轻转动凝胶染色和脱色的等效摇床
  5. 新不伦瑞克省I26孵育振荡器(Eppendorf,New Brunswick TM,型号:I26,目录号:M1324-0008)或可保持37℃和180rpm的等效振荡培养箱
  6. Heraeus Multifuge X1R离心机系列(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus TM Multifuge TM X1 /目录号:75004251)或具有能够容纳400毫升瓶子的转子的等效冷冻离心机并施加13,000克离心力的xg
  7. 通用水浴(VWR,型号:VWR通用水浴,目录号:89501-460)或能保持37°C的等效物
  8. 超声波清洗机 S-450模拟超声波仪(Emerson,Branson,型号:S-450,目录号:101-063-198)或等效超声波均质器,工作频率为20 kHz,尖端直径为3.2 mm
  9. 30毫升烧杯(康宁,PYREX ®,目录号:1000-30)或等效物
  10. 数字干式加热器(VWR,目录号:12621-088)或能够加热高达95°C的等效块式加热器
  11. 磁珠分离架(Thermo Scientific,目录号:MR02)或能够收集磁珠的等效机架
  12. 高压灭菌器(Getinge)或相当的高压灭菌器能够在施加15 atm压力的同时加热到121°C
  13. 量筒(100毫升)(VWR,目录号:65000-006)或等效的
  14. 量筒(1L)(VWR,目录号:65000-012)或等效的
  15. 介质/储存瓶(1升)(VWR,目录号:10754-820)或等效物
  16. 介质/储存瓶(100ml)(VWR,目录号:10754-814)或等效物
  17. 介质/储存瓶(500毫升)(VWR,目录号:10754-818)或等效物
  18. Ultrospec TM 10细胞密度计(GE Healthcare,目录号:80-2116-30)或等离子分光光度计,可以测量600 nm悬浮液中细胞的密度
  19. Mini-PROTEAN四细胞(Bio-Rad Laboratories,型号:Mini-PROTEAN Tetra Cell,目录号:1658004EDU)或等效的垂直电泳系统
  20. pH计Accumet TM AB15 plus basic(Fisher Scientific,型号:Fisher Scientific TM accumet TM> AB15 + Basic and BioBasic >,目录号:13-636-AB15P)或等效pH计,范围为0.000-10.000

程序

  1. 第一天制备培养基,试剂和单菌落
    1. 在250ml瓶中(用于开始过夜培养)和1L BHI培养液在2L锥形瓶中进行100ml大脑心脏输注(BHI)肉汤(用于大规模繁殖和蛋白质纯化)(参见配方1 )。
    2. 准备食谱1中列出的抗生素。
    3. 准备实验所需的所有库存解决方案(参见配方2)。
    4. 连续冷冻库存的。表皮炎 LM1680轴承 / Csm2 6HN (或金黄色葡萄球菌 RN4220轴承p crispr / Csm2 <6h>在含有10μg/ ml氯霉素的BHI琼脂平板上。如果是。使用表皮炎,补充15μg/ ml新霉素的培养基(见注1)。

  2. 第二天夜间文化准备
    1. 将10ml BHI培养液转入50ml锥形管中
    2. 加入10μl10mg / ml氯霉素,以选择p crispr / Csm2 6HN 。如果是。表皮霉素正在使用LM1680,另加10μl15 mg / ml新霉素。如果使用其他菌株和质粒,则酌情补充培养基与抗生素(见注1)
    3. 用无菌移液器吸头,挑选一个单一的菌落。表皮炎 LM1680轴承 / Csm2 6HN (或金黄色葡萄球菌 RN4220轴承p crispr / Csm2 6HN )。
    4. 将菌落重新悬浮在含有适当抗生素的10毫升高压灭菌培养基中
    5. 关闭松散的包含高压灭菌培养基,抗生素和感兴趣的菌落的50ml锥形管
    6. 将细菌培养物在设定为180rpm的摇动培养箱中于37℃孵育16-20小时
  3. 第3天。过度表达Cas10-Csm复合物的细胞的大规模繁殖
    1. 加入2L三角烧瓶中制备的1L BHI中,加入1ml 10mg / ml氯霉素。如果是。表皮霉素正在使用LM1680,另加1 ml 15 mg / ml新霉素。
    2. 接种1 L BHI加抗生素与第2天制备的整个10ml过夜培养物。
      注意:表皮葡萄球菌和金黄色葡萄球菌培养物的OD600 nm约为5-6。
    3. 用细胞密度计测量稀释培养物的初始OD600nm,以便于估计达到最终OD600nm所需的时间(见下文)。
    4. 在设定为180rpm的摇动培养箱中在37℃下培养细菌培养物。
    5. 让细菌培养物生长直到OD600nm = 2.00(如果从新鲜的过夜培养开始,则初始OD600nm = 0.05),约7-9小时。
    6. 将1升液体培养物分配到四个400毫升聚丙烯瓶中
    7. 以5,000×g离心细胞,4℃10分钟。
    8. 丢弃上清液,并用冰冷的蒸馏H 2 O(dH 2 O)的冰悬浮每个沉淀物。
    9. 将细胞悬浮液从所有小丸混合到干净的50ml锥形管中(冰上),并以5,000xg离心4℃,10分钟。
    10. 弃去上清液并将沉淀的细胞储存在-80°C(用于最佳纯化一周后)或直接进行纯化。

  4. 第4天净化 - 第1部分
    1. 在冰上解冻细胞沉淀1小时。
    2. 准备食谱3中描述的所有缓冲液。
    3. 将解冻的沉淀物重新悬浮在9ml冰冷的dH 2 O中
    4. 加入200μl1M MgCl 2和125μl新解冻的溶葡萄球菌素(2mg / ml储备液)。
    5. 在37℃水浴中孵育细胞悬浮液1小时。孵育30分钟后将管反转数次,以使悬浮液均质化
    6. 向裂解的细胞中加入10 ml再悬浮缓冲液,倒置管数次。细胞裂解物将变得非常粘稠(参见图2)

      图2.超声处理之前和之后的细胞裂解物的粘度。表皮细胞表达p / csm2 <6h>的LM1680细胞在超声处理之前(左),细胞裂解物是乳白色的,具有粘液的稠度。超声处理(右)后,细胞裂解物变成褐色,水分浓稠
    重要提示:所有后续步骤都应在冰上或4°C下进行
    1. 将Sonifier S-450调整至功率级6.0,占空比百分比调整为6.
      注意:这些设置对应于60 W功率输出,每秒固定重复频率为1个脉冲。 “6”的占空比表示每1秒脉冲(剩下的每秒40%)将发送60%的功率。
    2. 将裂解液倒入冰桶中的30 ml烧杯中。细胞裂解液应占据烧杯体积的一半,使其内的裂解物至少有1英寸的深度。
    3. 将超声波尖端浸入深度为1/2英寸的裂解液中,使得从烧杯底部保留约1/2英寸的间隙。
      重要提示:提示不要碰触烧杯的两侧或底部。
    4. 一次在冰上超声处理裂解物30秒,间隔一分钟。该循环应重复三次,或直到裂解物具有更多的水分稠度。通常,裂解液也会获得棕色色调(见图2)
    5. 将裂解液倒入50 ml锥形管中,以10,000 x g离心20分钟,4℃。离心后澄清的裂解物通常保留棕色色调(见图3)

      图3.第一次离心后超声处理的细胞裂解物的外观。细胞碎片沉淀后,澄清的裂解物保留黄棕色色调。

    6. 将上清液倒入新鲜的50ml锥形管中,以13,000 x g离心20分钟,4℃。
      注意:可以在SDS-PAGE上保存细胞沉淀并检查Cas10-Csm复合物的存在以评估其溶解度。
    7. 在两个旋转期间,准备/包装柱(要在冷藏室或熟食店冰箱内进行):
      1. 将1ml浆料Ni-NTA树脂吸入一次性重力流动柱中,并允许液体流过。
      2. 通过在4℃下将10毫升平衡缓冲液通过柱平衡Ni-NTA树脂
      3. 一旦大部分缓冲液通过,并且〜2-3毫米的缓冲液保留在树脂顶部。
    8. 最终旋转后(步骤D12),将裂解物通过0.22μmPES膜真空过滤器,并将滤液吸入无菌瓶中。
    9. 将经过滤的裂解物通过填充的Ni-NTA柱(在步骤D13中制备),并将其通过标示为“裂解液流通”的50ml锥形管中收集。
      注意:如果复合物怀疑已经通过柱而没有结合,则可以在SDS-PAGE上检查裂解物流通是否存在Cas10-Csm复合物。
    10. 通过将10ml洗涤1缓冲液通过柱洗涤柱。
    11. 收集洗涤1流通到一个15毫升锥形管 注意:如果复合体在洗涤期间怀疑已经通过色谱柱,则可以保存并检查洗涤1流通通道。* / em>
    12. 通过将10ml洗涤2缓冲液通过色谱柱第二次洗涤。
    13. 收集洗涤2流通到一个15毫升锥形管 注意:洗涤2流通可能会被保存,并在以后检查Cas10-Csm复合物,以防复合体在洗涤过程中怀疑已经通过色谱柱。
    14. 用5-7等分的洗脱缓冲液(每个馏分500μl)从柱洗脱蛋白质。当每个等分试样流经时,将其捕获到新鲜的适当标记的微量离心管中
    15. 加入新鲜标记的微量离心管中,将每个级分的20μl与5μl5x蛋白质加载缓冲液结合
    16. 在加热块上在95℃热样品5分钟。
    17. 将样品与蛋白质标准一起载入12%SDS-PAGE凝胶。
    18. 在Tris-glycine-SDS 1x缓冲液中以120V运行凝胶1小时
    19. 用考马斯蓝溶液染色凝胶10分钟,用脱色溶液脱色长达40分钟。
    20. 在白光下形成凝胶(图4)
    21. 在-20°C保存所需的洗脱液(见注2)。
      注意:避免复合物的反复冻融循环,因为这会导致蛋白质聚集。如果需要,将所需的洗脱液等分成较小的体积(〜100μl)。未经重复解冻的蛋白质复合物可以在-20℃下在洗脱缓冲液中储存至一年,而不会明显降解蛋白质或crRNA。


      图4.从S纯化后Cas10-Csm复合物的洗脱液。表达的表达 LM1680表达p / Csm2 <6h> 。显示从镍 - 琼脂糖柱收集的七个级分(E1-E7)在12%SDS PAGE凝胶上。蛋白质用考马斯蓝G-250染色。

  5. 第5天净化 - 第2部分
    1. 将10μlSera-Mag TM磁性链亲和素包被的小珠吸入微量离心管。
      注意:使用前,将珠悬浮液很好地上下移动多次。
    2. 用100μl1x退火缓冲液重新悬浮珠子(参见配方2)。
      注意:一旦溶液变成均匀的棕色,就可以实现珠重新悬浮,并且在管的侧面或底部没有看到珠积聚。当管从磁性架上取出时,其内部的珠可以很容易地重新悬浮。
    3. 通过将微量离心管放入磁性分离架中并等待珠子收集在管子的侧面,从而将珠子打碎。
      注意:根据使用的珠子和机架,收集过程可能在30秒到2分钟之间变化。收集过程将在上清液变清晰后完成,并且珠(褐色)在磁铁所在管侧聚集。如果珠子围绕管的一侧广泛分布,将微量离心管顺时针旋转几度,或者在机架内逆时针旋转,以将所有的珠子更靠近在一起。
    4. 轻轻移出上清液,不要打扰珠子。
    5. 重复洗涤(步骤E2-E4)三次。
    6. 收集最浓缩的Cas10-Csm复合物级分(在第4天获得),并与2ng与crRNA反义的5'生物素化寡核苷酸(参见注3)结合。
    7. 让混合物在室温下孵育30分钟。
    8. 将含有珠的微量离心管从磁性架上取出,并将Cas10-Csm复合物/寡聚混合物加入平衡的链霉亲和素珠中。
    9. 重新悬浮珠,直到混合物显得均匀,并使其在室温退火30分钟
    10. 通过将微量离心管放回磁架〜30秒,将珠收集在管的侧面
    11. 轻轻移出上清液,不要打扰珠子。
    12. 从磁性架上取出管,并将珠子悬浮在100μl1x退火缓冲液中
    13. 通过将微量离心管放回磁架〜30秒,将珠收集在管的侧面
    14. 轻轻移出上清液,不要打扰珠子。
    15. 重复洗涤(步骤E12-E14)两次。
    16. 向珠中加入20μl1x退火缓冲液,加入5μl5x蛋白加载缓冲液。
    17. 将样品在95℃加热块加热5分钟
    18. 将管放回磁性架,以从溶液中除去珠子。
    19. 将蛋白质标准物上清液加入12%SDS-PAGE凝胶中
    20. 在Tris-glycine-SDS 1x缓冲液中以120V运行凝胶1小时
    21. 用考马斯蓝溶液染色凝胶10分钟,用脱色溶液脱色长达40分钟。
    22. 在白光下形成凝胶(图5)

      图5. 表皮霉素用生物素化寡核苷酸进行亲和纯化后,从磁性链亲和素珠中收集Cas10-Csm复合物,使其与反向 crRNAs 蛋白质在12%SDS PAGE凝胶上分离,与考马斯蓝色G250。

数据分析

该测定用作评估Cas10-Csm复合物完整性的定性测量。 SDS-PAGE凝胶上特定蛋白质亚基的存在或不存在将指示其是否与复合物稳定相关。使用该方案,可以通过从第一纯化过程获得的Cas10-Csm复合物等分试样中直接提取crRNA,然后用γ- 32'进行5'末端标记来确定复合物内crRNA的存在或不存在和长度, / sup> P-ATP,并将其分解在8-12%的聚丙烯酰胺凝胶上。在确定复杂的稳定性之前,这些测定必须在三次独立的重复中给出一致的结果。对于首次表征的复合物,必须使用蛋白质印迹法确认凝胶中每个亚基的身份。这些附加方法在(Hatoum-Aslan等人,2013)中描述)。

笔记

  1. 该方案描述了来自S的Cas10-Csm复合物的纯化。表皮炎 LM1680或 S。金黄色葡萄球菌携带p / / / / / / / / / / / / / / />的RN4220细胞是从S编码整个III-A型CRISPR-Cas系统的质粒。具有引入到csm2的N末端的6x-His标签的表皮细胞RP62a(Hatoum-Aslan等人,2013)。 S上。表皮炎 LM1680是一种衍生物。具有包含CRISPR-Cas基因座(Hatoum-Aslan等人,2013)的染色体缺失的表皮葡萄球菌RP62a。 S上。金黄色葡萄球菌 RN4220是用作克隆中间体的 crispr -strain(Nair等人,2011)。当在p crispr / Csm2 H6N 上重新引入时,CRISPR-cas系统在两个菌株中保留完整的功能。 p crispr / Csm2 H6N 需要在10μg/ ml氯霉素中进行选择。另外,使用15μg/ ml的新霉素专门用于选择S。表皮炎 RP62a或LM1680。该方案也可以适应于在不同质粒或不同菌株中过表达CRISPR-Cas系统,然而,抗生素选择也必须相应地进行修改。如果使用野生型菌株,在理论上过表达编码CRISPR-Cas系统的一个亚基6x-His的质粒,即使在背景系统的染色体拷贝存在的情况下也可以使Cas10-Csm复合物纯化。
  2. 可以在第一次纯化后进行质谱分析,以检测与复合物松散相关的潜在结合配偶体。此外,复合物中crRNA的存在或不存在及其长度可以通过在第一次纯化后终止标记RNA并在尿素PAGE凝胶上拆分来从复合物中提取RNA来确定。如果需要更清洁的制剂或评估复杂的稳定性,则应进行第5天描述的第二次纯化。
  3. 在该实施例中使用的生物素化的寡核苷酸的序列与重复 - 间隔物阵列的第一间隔物( spc 1 1)互补:
    5'-Biotin-TEG-ACGAGAACACGUAUGCCGAAGUAUAUAAAUC

食谱

  1. 媒体和抗生素
    1. 脑心脏输液(BHI)肉汤
      1. 溶解3.7 g / 100ml dH 2 O - / -
      2. 在121℃高压灭菌30分钟
      3. 在室温下存放
    2. 氯霉素储备液(10mg / ml)
      1. 将100mg氯霉素溶解于10ml的100%乙醇中
      2. 通过0.22μm注射器过滤器
      3. 储存于4°C
    3. 新霉素原料(15mg / ml)
      1. 将150毫克新霉素溶解于10ml dH 2 O - / -
      2. 通过0.22μm注射器过滤器
      3. 储存于4°C
  2. 库存解决方案
    1. 1M MgCl 2
      1. 将9.521 g MgCl 2放入100 ml量筒中
      2. 填充dH 2 O达100 ml
      3. 在室温下存放在瓶子里
    2. 1M Tris-HCl,pH 8.3
      1. 将121.14 g Tris放入1升量筒中
      2. 用800毫升dH 2 O溶液溶解
      3. 用12N HCl将pH调节至8.3
      4. 填充dH 2 O至1 L
      5. 在室温下存放在瓶子里
    3. 1M Tris-HCl,pH6.8
      1. 将121.14 g Tris放入1升量筒中
      2. 用800毫升dH 2 O溶液溶解
      3. 用12N HCl将pH调节至6.8
      4. 填充dH 2 O至1 L
      5. 在室温下存放在瓶子里
    4. 1 M KCl
      1. 将7.455克KCl放入100毫升量筒中
      2. 填充dH 2 O达100 ml
      3. 在室温下存放在瓶子里
    5. 500mM EDTA,pH 8.0
      1. 将93.06g EDTA,二钠盐,二水合物(MW = 372.24g / mol)放入500ml烧杯中
      2. 尽可能多地用350ml dH 2 O - / - 溶解
      3. 用10N NaOH调节pH至8.0,同时不断搅拌 注意:ED TA不会完全溶解,除非其pH为8.0。然而,当 时,EDTA的pH持续下降。因此,必须使用不断添加10 N NaOH以使EDTA完全溶解,最终pH为8.0。
      4. 填充dH <2> O至500 ml
      5. 在室温下存放在瓶子里
    6. 10%十二烷基硫酸钠(SDS)
      1. 将1克SDS放入15毫升锥形管中
      2. 用6ml dH 2 O O溶解
      3. 填充dH <2> O至10 ml
      4. 在室温下存放

        由于粉状SDS会引起严重的眼睛和肺部损伤,可购买20%的溶液(VWR Cat。 97062-440),并稀释与dH <2> O(1:1)。


    7. 1%溴酚蓝
      1. 将0.1g溴酚蓝放入15 ml锥形管中
      2. 填充dH <2> O至10 ml
      3. 在室温下存放
    8. 2mg / ml重组溶葡萄球菌素
      1. 将50mg溶葡萄球菌溶解在25ml NaOAc中,pH4.5
      2. 将0.5ml等分试样分配到微量离心管中
      3. 储存于-80°C
      4. 解冻之前使用
    9. 2x裂解缓冲液
      1. 将11.998g NaH 2 PO 4置于1L量筒([最终] = 100mM)中)/ /
      2. 将35.064g NaCl放入量筒([最终] = 600mM)
      3. 用dH 2 O填充至800ml
      4. 用10N NaOH调节pH至8.0
      5. 填写1 L
      6. 在室温下存放
    10. 5x退火缓冲液
      1. 将2.5毫升1M Tris-HCl,pH 8.3放入100毫升瓶中([最终] = 25毫米)
      2. 将37.5ml 1M KCl放入瓶中([最终] = 375mM)
      3. 将1ml 500mM EDTA,pH8.0放入瓶中([最终] = 5mM)
      4. 加入59毫升dH 2 O - / -
      5. 在室温下储存瓶子
    11. 5倍蛋白加载缓冲液
      1. 将0.5ml 1M Tris-HCl,pH 6.8放入15ml锥形管中([最终] = 62.5mM)
      2. 将0.8ml 100%甘油置入管中([最终] = 10%)
      3. 将1.6ml 10%SDS溶液放入管中([最终] = 2%)
      4. 加入0.4ml 14.3Mβ-巯基乙醇([最终] = 715mM)
      5. 向管中加入0.4ml的1%溴酚蓝([最终] = 0.05%)
      6. 加入4.3ml的dH 2 O - / -
      7. 将1ml等分试样分配到微量离心管中
      8. 储存于-20°C
    12. 考马斯蓝染色溶液
      1. 将1克考马斯蓝G-250放入1升量筒([最终] = 0.1%)
      2. 将500毫升甲醇放入量筒([最终] = 50%)
      3. 将100ml乙酸放入量筒([最终] = 10%)
      4. 将400ml dH 2 O加入量筒([最终] = 40%)
      5. 在室温下存放在瓶子里
    13. 解决问题
      1. 将500毫升甲醇放入1升量筒([最终] = 50%)
      2. 将100毫升乙酸放入量筒([最终] = 10%)
      3. 将400ml dH 2 O加入量筒([最终] = 40%)
      4. 在室温下存放在瓶子里
    14. 1×Tris-甘氨酸-SDS缓冲液
      1. 将100毫升10倍Tris-甘氨酸-SDS缓冲液放入1升量筒中
      2. 填充dH 2 O至1 L
      3. 在室温下存放在瓶子里
  3. 缓冲液在第4天准备净化 - 第1部分
    注意:所有以下缓冲区应该新鲜准备。保持缓冲区在冰上。从1升培养物中准备10ml每个细胞沉淀的缓冲液。
    1. 洗脱缓冲液(10ml [最终体积])
      1. 将60mg NaH 2 PO 4置于50ml锥形管中([最终] = 50mM)
      2. 加入175mg NaCl([最终] = 300mM)
      3. 加入170mg咪唑([最终] = 250mM)
      4. 加入1ml甘油([最终] = 10%)
      5. 加入8 ml dH 2 O - / -
      6. 用10N NaOH调节pH至8.0
      7. 使用dH O
        将最终体积加至10ml
    2. 重悬浮缓冲液(10ml [最终体积])
      1. 将9.2ml 2x裂解缓冲液放入50ml锥形管中
      2. 加入800μl洗脱缓冲液
      3. 加入1个完整的片剂蛋白酶抑制剂(游离EDTA)
      4. 盖管紧紧旋转直到片剂完全溶解(30秒-1分钟)
      5. 加入10μlTriton X-100([最终] = 0.1%)
      6. 轻轻反转管,直到Triton X-100完全溶解
    3. 平衡缓冲液(10ml [终体积])
      1. 将5 ml 2x裂解缓冲液放入50 ml锥形管中
      2. 加入5ml dH O O
    4. 洗1缓冲液(10ml [终体积])
      1. 将5 ml 2x裂解缓冲液放入50 ml锥形管中
      2. 加入800μl洗脱缓冲液
      3. 加入4.2ml dH 2 O
    5. 洗2缓冲液(10ml [最终体积])
      1. 将5 ml 2x裂解缓冲液放入50 ml锥形管中
      2. 加入800μl洗脱缓冲液
      3. 加入3.2ml dH 2 O O
      4. 加入1ml甘油([最终] = 10%)

致谢

A.H-A。由阿拉巴马大学(UA)艺术与科学学院支持; UA学院研究,奖学金和创意活动研究所(CARSCA)的资助;和国立卫生研究院[5K22AI113106-02]。该方案改编自于Hatoum-Aslan等人,J.Biol Chem。,2013年出版的。

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chou-Zheng, L. and Hatoum-Aslan, A. (2017). Expression and Purification of the Cas10-Csm Complex from Staphylococci. Bio-protocol 7(11): e2353. DOI: 10.21769/BioProtoc.2353.
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